Lentivirus Vaccine Based On The Recombinant Viral Vaccine Against Yellow Fever

Abstract

The present invention relates to an attenuated, recombinant viral vaccine against yellow fever which expresses heterologous sequences of a lentivirus and is used as an immunisation agent to induce an immune response to lentivirus.

Description

FIELD OF THE INVENTION

[0002] The present invention relates to the development of a vaccine against lentivirus, and more specifically against primate lentiviruses such as HIV (Human Immunodeficiency Virus) and SIV (Simian Immunodeficiency Virus).

[0003] The present invention concerns the use of attenuated recombinant yellow fever vaccine virus 17D for expression of HIV and SIV antigens for immunization. The invention provides immunization schemes using yellow fever virus expressing HIV or SIV antigens. For example, the recombinant virus may be used in a "prime-boost" protocol immunizing an individual with a recombinant polynucleotide or recombinant Bacillus Calmette-Guerin (rBCG) vaccine, which expresses one or more HIV or SIV antigens, followed by booster dose with recombinant Yellow Fever virus 17D, which expresses one or more HIV or SIV antigens.

BACKGROUND OF THE ART

[0004] Over the 28-year period since the scientists identified the human immunodeficiency virus (HIV) as the cause of the acquired immunodeficiency syndrome (AIDS), the virus has spread inexorably, giving rise to one of the most devastating pandemics ever recorded in history. More than 20 million people have already died of AIDS and about 33 million people are living with HIV infection.

[0005] However, none of the vaccine regimens tested in HIV vaccine efficacy trials to date has reduced HIV infection or replication rates.

[0006] Structural aspects and the enormous variability of the Envelope glycoprotein have frustrated the efforts to broadly induce reactive neutralizing antibodies against HIV [1]. Investigators have therefore focused their attention on T-cell-based vaccines. The recent trial of a recombinant adenovirus[Ad5]-vectored vaccine was widely seen as an important test of this concept [3,4]. Unfortunately, vaccinees became infected at higher rates than control groups [3].

[0007] Several studies have shown the key role of CD+8 T cells in the control of HIV infections as well as of simian immunodeficiency virus (SIV) infections. As a result, various modalities of response-inducing vaccines regarding CD8+ T-cell effector response have been currently investigated and developed [5, 6, 7, 8].

[0008] Hence, it is also desirable that new approaches are developed in order to reduce the incidence of HIV infection or improve the consequences of the infection. As regards this aspect, vaccines are particularly relevant.

SUMMARY OF THE INVENTION

[0009] The technical matter described herein relates to pharmaceutical compositions that may be used as immunogenic compounds or vaccines against lentivirus, and more specifically as immunogenic compounds or vaccines against HIV (Human Immunodeficiency Virus) or SIV (Simian Immunodeficiency Virus).

BRIEF DESCRIPTION OF THE FIGURES

[0010] FIG. 1 shows viral growth curves in Vero cells.

[0011] FIG. 2 shows that YF17D replicates and induces neutralizing antibodies, virus-specific CD8+ T cells, and CD8+ T-cell activation in rhesus monkeys.

[0012] FIG. 3 shows the study of infection and immunogenicity of YF17D/SIVGag45-269 virus in rhesus monkeys.

[0013] FIG. 4 shows that vaccination with rYF17D/SIVGag45-269 yielded a robust expansion of Gag-specific responses in a monkey initially vaccinated with rBCG.

[0014] FIG. 5 shows that the result of the vaccination with rYF17D/SIVGag45-269 of r01056 [sic].

DETAILED DESCRIPTION OF THE INVENTION

[0015] The described compositions typically include a recombinant polynucleotide or a panel of recombinant polynucleotides. Adequate HIV or SIV polynucleotides may encode at least one fragment of a HIV or SIV polypeptide including Gag, Pol, Rev, Tat, Nef, Vif, Vpx, Vpr, Env, Vpu, [sic] (preferably at least one fragment of Gag, Vif, and Nef). The recombinant polynucleotide or panel of recombinant polynucleotides may be present in a recombinant virus or in a panel of recombinant viruses which express polypeptides encoded by the recombinant polynucleotides. The disclosed compositions may be used in methods for inducing immune response against HIV, which may include HIV-1 or HIV-2, or SIV (for example, response based on cytotoxic T cells (CTC), and, optionally, humoral or antibody-based response. [sic]

[0016] Disclosed compositions may include a mixture of recombinant polynucleotides, which may be present in a viral vector or in other nucleotide vector such as a plasmid or a bifunctional vector ("shuttle" vectors). Typically, the polynucleotides are present in expression vectors to express the polypeptides encoded by the polynucleotides. Adequate vectors may include but are not limited to viral vectors (for example, attenuated recombinant viruses). The pharmaceutical composition may include free recombinant nucleic acid or recombinant nucleic acid packaged into one or more virus particles (for example, replication-defective viral particles or attenuated virus). For instance, the recombinant polynucleotides contemplated herein may be present in one or more recombinant viruses (for example, recombinant yellow fever viruses) and the compositions may include a mixture of recombinant viruses.

[0017] The compositions may include a polynucleotide or a panel of polynucleotides (for example, at least about 2-9 polynucleotides) present in one or more expression vectors (for example, a viral vector or other expression vector, [sic] which encode the complete Gag polypeptide or a fragment/part of it. Optionally, the compositions may include a polynucleotide or a panel of polynucleotides present in one or more expression vectors which encode the complete Vif polypeptide or a fragment/part of it. Optionally, the compositions may include a polynucleotide or a panel of polynucleotides present in one or more expression vectors which encode the complete Nef polypeptide or a fragment/part of it. Optionally, the compositions may include a polynucleotide or a panel of polynucleotides present in one or more expression vectors which encode the complete Tat polypeptide or a fragment/part of it. Optionally, the compositions may include a polynucleotide or a panel of polynucleotides present in one or more expression vectors which encode Rev polypeptide or a fragment/part of it. Optionally, the compositions may include a polynucleotide or a panel of polynucleotides present in one or more expression vectors which encode the complete Pol polypeptide or a fragment/part of it. Optionally, the compositions may include a polynucleotide or a panel of polynucleotides present in one or more expression vectors which encode the complete Vpr polypeptide or a fragment/part of it. Optionally, the compositions may include a polynucleotide or a panel of polynucleotides present in one or more expression vectors which encode the complete Vpx polypeptide or a fragment/part of it. The polynucleotides or panels of polynucleotides may encode different polypeptides or different fragments of polypeptides. The encoded polypeptides or fragments of polypeptides may overlap (for example, in about 9-20 amino acids). The fragments are typically in the length range of 5-100 amino acids (or from 5-50, 10-50, 10-25, 5-15, or 10-15 amino acids) and include at least one epitope of the polypeptide from which the fragment is derived. In some embodiments, a fragment may include an N-terminal amino acid which is not normally present in the complete polypeptide from which the fragment is derived.

[0018] The pharmaceutical composition may comprise an effective amount or concentration of recombinant polynucleotides or recombinant viruses to induce a protective or therapeutic immune response against HIV infection in humans or SIV infection in simians. Inducing a protective or therapeutic immune response may include the potentiation of a CD8+ and/or CD4+ response to one or more HIV or SIV epitopes. Inducing a protective response may include the induction of sterilizing immunity against HIV or SIV. Inducing a therapeutic response may include the reduction of the viral load of an individual, for example, as determined by the measurement of the amount of circulating virus before and after delivering the composition. In some embodiments, the viral load is reduced to less than about 10,000 vRNA copies/mL (preferably to less than about 5,000 vRNA copies/mL, more preferably to less than about 2000 vRNA copies/mL, much more preferably to less than about 1500 vRNA copies/mL, and even more preferably to less than about 1000 vRNA copies/mL) at least about 3 months after the composition administration. The induction of a therapeutic response may include the increase in CD4+ T-cell count of the individual (for example, at least a twofold, threefold, or fourfold increase) at least 3 months after the composition administration.

[0019] Methods inducing protective or therapeutic immune responses against HIV or SIV infection through the administration of the pharmaceutical compositions described herein (for example, as immunogenic compounds or vaccines) to an individual who needs them, which may include a human who has already acquired or is at risk of acquiring a lentivirus infection, are also disclosed.

[0020] As used herein, a "nucleic acid vaccine" or "free DNA vaccine" relates to a vaccine which includes one or more expression vectors which encode B-cell and/or T-cell epitopes and provide an immunoprotective response to the individual who is being vaccinated. Nucleic acid vaccines as defined here, which may include expression vector plasmids, are not typically encapsidated into a viral particle. The vaccine nucleic acid is directly introduced into the cells of the individual who is subjected to the vaccinal regimen. This approach is described, for example, in U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based administration technologies may include pure DNA ("naked DNA") administration, facilitated distribution (for example, through the use of bupivacaine, polymers, or peptides), and administration in the presence of cationic lipid complex or liposomes. Nucleic acids may be delivered using ballistic administration (for example, as described in U.S. Pat. No. 5,204,253), by pressure (for example, as described in U.S. Pat. No. 5,922,687), or by electroporation.

[0021] The nucleic acids used in the compositions and vaccines disclosed herein may encode one or more viral proteins or portions of one or more viral proteins. In some embodiments, the nucleic acids may encode an epitope (for example, a T-cell epitope) which is not part of a known HIV or SIV functional protein or which is encoded by a HIV or SIV alternative open reading frame (for example, a "cryptic" epitope). (See, for example, Maness et al., J. Exp. Med. (2007), 204:2505-12; and Cardinaud et al., J. Exp. Med. (2004), 199:1053-1063, which are all incorporated by reference herein in their entireties). A cryptic epitope may be located in an open reading frame (ORF) whose translation is not typically observed in the course of HIV or SIV viral replication. For example, Maness et al. discloses a cryptic epitope in SIVmac239 called "cRW9", which is located in the +2 reading frame relative to the ORF encoding the envelope protein. This cryptic epitope is located in the same ORF that encodes exon 1 of the Rev protein, but it is downstream of the only known splice donor site and so is not predicted to be translated under "normal" biological circumstances.

[0022] The pharmaceutical compositions disclosed herein may be formulated as vaccines for administration to an individual who needs them. These compositions may be formulated and/or administered at dosages and through techniques that are well known by physicians, taking into account factors such as age, gender, weight, particular conditions of the patient, and route of administration. The compositions may include pharmaceutical carriers, diluents, or excipients as they are known [in the art]. Moreover, the compositions may include preservatives (for example, antimicrobial or antibacterial agents such as benzalkonium chloride) or adjuvants.

[0023] The pharmaceutical compositions may be administered prophylactically or therapeutically. In prophylactic administration, vaccines may be administered in a sufficient amount to induce CD8+, CD4+, and/or antibody responses for protection against infection. In therapeutic applications, vaccines are administered to a patient in a sufficient amount to induce a therapeutic effect (for example, CD8+, CD4+, and/or antibody responses for HIV or SIV antigens or epitopes encoded by the polynucleotides of the composition [sic], which heals or at least partially suspends or delays the symptoms and/or complications of HIV or SIV infection (i.e., a "therapeutically effective dose").

[0024] The compositions disclosed herein may include recombinant polynucleotides or recombinant viruses which encode and express complete HIV or SIV polypeptides or their fragments. As used herein, a "minigene" encodes only part of a gene. For example, a minigene for HIV Gag may encode only 5-100 amino acids of the Gag polypeptide (or 5-50, 10-50, 5-25, 10-25, 5-15, or 10-15 amino acids). Minigenes which encode only part of a gene are described in the U.S. patent application Ser. No. 12/022,530, filed on Jan. 30, 2008, which claims the benefit of U.S. Provisional Application No. 60/898,644, filed on Jan. 31, 2007, the contents of which are incorporated by reference herein in their entireties. For example, a minigene for HIV Gag may encode a Gag fragment which includes only 5-100 amino acids of the Gag polypeptide (or 5-50, 10-50, 5-25, 10-25, 5-15, or 10-15 amino acids). In some embodiments, the recombinant polynucleotide or recombinant viruses may encode and express a SIV polypeptide fragment as disclosed in Table 1 or a corresponding HIV polypeptide fragment.

TABLE-US-00001 TABLE 1 Epitope-specific sequences of amino acids. MHC class I Protein Name Sequence restriction Tat SL8 STPESANL A*01 Nef IW9 IRYPKTFGW B*17 MW9 MHPAQTSQW B*17 YY9 YTSGPGIRY A*02 YY9* YTYEAYVRY A*02 AL11 ARRHRILDIYL B*08 ML10 MRRSRPSGDL B*08 RL10 RRRLTARGLL B*08 Vif HW8 HLEVQGYW B*17 WY8 WTDVTPNY A*02 RL8 RRDNRRGL B*08 RL9 RRAIRGEQL B*08 Ver KL9 KRLRLIHLL B*08 RL10 RRRWQQLLAL B*08 Pol p10 LV10 LGPHYTPKIV A*01 protease YL8 YHSNVKEL A*07 Env gp120 CL9 CAPPGYALL A*01 Env gp41 TL9 TVPWPNASL A*01 Env gp41 RY8 RTLLSRVY A*02 Env gp41 FW9 FHEAVQAVW B*17 Gag p17 GY9 GSENLKSLY A*02 matrix CM9 CTPYDINQM A*01 Gag p27 QI9 QNPIPVGNI A*01 capsid LF8 LAPVPIPF A*01 Gag p27 RW9 RAPRRQGCW B*17 capsid Vpx II11 IPPGNSGEETI A*01

[0025] In some embodiments, the recombinant polynucleotide or recombinant viruses may comprise a "minigene" of SIV or HIV and express a SIV polypeptide fragment or a corresponding HIV polypeptide fragment as disclosed in Tables 2-6.

TABLE-US-00002 TABLE 2 SIV and HIV Gag Protein Fragments Protein Sequence 1(a) SVLSGKKADELEKIRLRPNGKKKYMLKHVVWAANELDRFGLAESLLE SIV Gag p17 1(b) SVLSGGKLDKWEKIRLRPGGKKTYQLKHIVWASRELERFAVNPGLLE HIV Gag p17 2(a) FGLAESLLENKEGCQKILSVLAPLVPTGSENLKSLYNTVDV SIV Gag p17 2(b) FAVNPGLLETGGGCKQILVQLQPSLQTGSEELKSLYNAVAT HIV Gag p17 3(a) NKEGCQKILSVLAPLVPTGSENLKSLYNTVCVIWCIHAEEKVKHTEEA SIV Gag p17 KQIVQRHLVVETGTTETMPKTSR 3(b) TGGGCKQILVQLQPSLQTGSEELKSLYNAVATLYCVHQGIEVRDTKEA HIV Gag p17 LDKIEEEQNKSKKKAQQAAA 4(a) GNYVHLPLSPRTLNAWVKLIEEKKFGAEVVPGFQALSEGCTPYDI SIV Gag p27 4(b) GQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDL HIV Gag p24 5(a) SEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAADWDLQHPQPAPQQ SIV Gag p27 GQLREPSGSDIAGTTSSVDEQIQWMYRQQNPIP 5(b) SEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPAHAGPN HIV Gag p24 APGQMREPRGSDIAGTTSTLQEQIGWMTSNPPVP 6(a) SEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAADWDLQHPQPAPQQ SIV Gag p27 GQ 6(b) SEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPAHAGPN HIV Gag p24 APGQ 7(a) MYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTNIPDVKQGPKEPFQSY SIV Gag p27 VDRFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGVNPTLE EMLTACQGVGGPGQKARLMAEALKEALAPVPIPFAAAQQRGPRKPIKC WNCGKEGHSARQCRAPRRQGCW 7(b) MTSNPPVPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYV HIV Gag p24 DRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEE MMTACQGVGGPSHKARILAEAMSQVTSPANIMMQRGNFRNQRKTIKCF NCGKEGHLARHCRAPRKKGCW 8(a) TTSSVDEQIQWMYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTNIPDV SIV Gag p27 KQGPKEPFQSYVDRFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLV LKGLGVNPTLEEMLTACQGVGGPG 8(b) TTSTLQEQIGWMTSNPPVPVGEIYKRWIILGLNKIVRMYSPVSILDIR HIV Gag p24 QGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTIL KALGPAATLEEMMTACQGVGGPS

TABLE-US-00003 TABLE 3 SIV and HIV Vif Protein Fragments 10(a) MEEEKRWIAVPTWRIPERLERWHSLIKYLKYKTKDLQKVCYVPHFKVGWA SIV Vif WWTCSRVIFPLQEGSHLEVQGYWHLTPEKGWLSTYAVRITWYSKNFWTDV TPNYADILLH 10(b) MENRWQVMIVWQVDRMRIKTWKSLVKHHMYISKKAKEWVYRHHYESTHPR HIV Vif ISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNTQVDP DLADKLIH 11(a) PNYADILLHSTYFPCFTAGEVRRAIRGEQLLSCCRFPRAHKYQVPSLQYL SIV Vif ALKVVSDVRSQGENPTWKQWRRDNRRGLRMAKQNSRGDKQRGGKPPTKGA NFPGLAKVLGILA 11(b) PDLADKLIHLHYFDCFSDSAIRHAILGHRVRPKCEYQAGHNKVGSLQYLA HIV Vif LTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH 12(a) MEEEKRWIAVPTWRIPERLERWHSLIKYLKYKTKDLQKVCYVPHFKVGWA SIV Vif WWTCSRVIFP 12(b) MENRWQVMIVWQVDRMRIKTWKSLVKHHMYISKKAKEWVYRHHYESTHPR HIV Vif ISSEVHIP 13(a) WWTCSRVIFPLQEGSHLEVQGYWHLTPEKGWLSTYAVRITWYSKNFWTDV SIV Vif TPNYADILLH 13(b) ESTHPRISSEVHIPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRY HIV Vif NTQVDPDLADKLIH 14(a) PNYADILLHSTYFPCFTAGEVRRAIRGEQLLSCCRFPRAHKYQVPSLQYL SIV Vif ALKVVSDVR 14(b) PDLADKLIHLHYFDCFSDSAIRHAILGHRVRPKCEYQAGHNKVGSLQYLA HIV Vif LTALITPKK 15(a) LALKVVSDVRSQGENPTWKQWRRDNRRGLRMAKQNSRGDKQRGGKPPTKG SIV Vif ANFPGLAKVLGILA 15(b) LALTALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH HIV Vif

TABLE-US-00004 TABLE 4 SIV and HIV Nef Protein Fragments 16(a) GLDKGLSSLSCEGQKYNQGQYMNTPWRNPAEEREKLAYRKQNMDDIDEED SIV Nef DDLVGVSVRPKVPLRTMSYKLAIDMSHFIKEKGGLEGIYYSARRHRILDI YLEKEEGIIPDWQDYTSGPGIRYPKTFGWLWKLVPVNVSDEAQEDEEHYL MHPAQTSQWDDPWGEV 16(b) MGGKWSKSSKAGWPQVREKIKQTPPAAEGVGAVSQDLDKHGAITSSNMNN HIV Nef ADCVWLQAQEDEEVGFPVRPQVPLRPMTFKGAFDLSFFLKEKGGLDGLIY SRKRQEILDLWVYNTQGFFPDWQNYTPGPGVRLPLCFGWCFKLVPVDPRE VEEDNKGENNCLLHPLSQHGMEDEHKEV 17(a) GLDKGLSSLSCEGQKYNQGQYMNTPWRNPAEEREKLAYRKQNMDDIDEED SIV Nef DD 17(b) MGGKWSKSSKAGWPQVREKIKQTPPAAEGVGAVSQDLDKHGAITSSNMNN HIV Nef ADCVWLQAQEDEE 18(a) DDIDEEDDDLVGVSVRPKVPLRTMSYKLAIDMSHFIKEKGGLEGIYYSAR SIV Nef RH 18(b) DCVWLQAQEDEEVGFPVRPQVPLRPMTFKGAFDLSFFLKEKGGLDGLIYS HIV Nef RKR 19(a) GIYYSARRHRILDIYLEKEEGIIPDWQDYTSGPGIRYPKTFGWLWKLVPV SIV Nef 19(b) GLIYSRKRQEILDLWVYNTQGFFPDWQNYTPGPGVRLPLCFGWCFKLVPV HIV Nef 20(a) GWLWKLVPVNVSDEAQEDEEHYLMHPAQTSQWDDPWGEV SIV Nef 20(b) GWCFKLVPVDPREVEEDNKGENNCLLHPLSQHGMEDEHKEV HIV Nef

TABLE-US-00005 TABLE 5 SIV and HIV Tat Protein Fragments 21(a) METPLREQENSLESSNERSSCISEADASTPESANLGEEILSQLYRPLEA SIV Tat 21(b) MEPVDPRLEPWKHPGSQPKTPCTKCYCKKCCLHCQVCFMTKGLGISYGRK HIV Tat 22(a) SQLYRPLEACYNTCYCKKCCYHCQFCFLKKGLGICYEQSRKRRRTPKKAK SIV Tat 22(b) SQPKTPCTKCYCKKCCLHCQVCFMTKGLGISYGRKKRRQRRRAPQDNKNH HIV Tat

TABLE-US-00006 TABLE 6 SIV and HIV Rev Protein Fragments 23(a) MSNHEREEELRKRLRLIHLLHQTNPYPTGPGTANQRRQRKRRWRRRWQQ SIV Rev 23(b) MAGRSGSTDEELLRAVRIIKILYQSNPYPSSEGTRQARRNRRRRWRARQR HIV Rev 24(a) RRWRRRWQQLLALADRIYSFPDPPTDTPLDLAIQQLQNLAIESIPDPPTN SIV Rev TPEALCDPTEDSRSPQD 24(b) RRNRRRRWRARQREICALSERILSSCLGRPTEPVPLPLPPLERLTLDCSE HIV Rev DCGTSGTQQSQGTETGVGRPQISGE

[0026] A minigene of SIV or a minigene of HIV may encode a polypeptide comprising a SIV or HIV polypeptide fragment and also comprise an N-terminal methionine. A minigene of SIV or a minigene of HIV may include codon sequences which are optimized for expression (for example, in animal, organism or recombinant virus as contemplated herein). Exemplary SIV minigenes including codon sequences which are optimized for expression by the yellow fever virus are depicted in Table 7.

TABLE-US-00007 TABLE 7 Optimized-Sequence Minigenes Name 1. SIVmac239Gag(6-52) DNA Seq. 5'- GTCGACAGGAGCCACCATGAGCGTGCTGAGCGGCAAGAAGGCCGACGAGC TGGAGAAGATCCGGCTGCGGCCCAACGGCAAGAAGAAATACATGCTGAAG CACGTGGTGTGGGCCGCCAACGAGCTGGACCGGTTCGGCCTGGCCGAGAG CCTGCTGGAGTGAGAATTCGCGGCCGC-3' AA Seq. MSVLSGKKADELEKIRLRPNGKKKYMLKHVVWAANELDRFGLAESLLE 2. SIVmac239Gag(44-84) DNA Seq. 5'- GTCGACAGGAGCCACCATGTTCGGCCTGGCCGAGAGCCTGCTGGAGAACA AGGAGGGCTGCCAGAAGATCCTGAGCGTGCTGGCCCCTCTGGTGCCCACC GGCAGCGAGAACCTGAAGAGCCTGTACAACACCGTGTGCGTGTGAGAATT CGCGGCCGC-3' AA Seq. MFGLAESLLENKEGCQKILSVLAPLVPTGSENLKSLYNTVCV 3. SIVmac239Gag(76-123) DNA Seq. 5'- GTCGACAGGAGCCACCATGAAGAGCCTGTACAACACCGTGTGCGTGATCT GGTGCATCCACGCCGAGGAGAAGGTGAAGCACACCGAGGAGGCCAAGCAG ATCGTGCAGCGGCACCTGGTGGTGGAGACCGGCACCACCGAGACCATGCC CAAGACCAGCCGCTGAGAATTCGCGGCCGC-3' AA Seq. MKSLYNTVCVIWCIHAEEKVKHTEEAKQIVQRHLVVETGTTETMPKTSR 4. SIVmac239Gag(142-186) DNA Seq. 5'- GTCGACAGGAGCCACCATGGGCAACTACGTGCACCTGCCCCTGAGCCCCC GGACCCTGAACGCCTGGGTGAAGCTGATCGAGGAGAAGAAGTTCGGCGCC GAGGTGGTGCCCGGCTTCCAAGCCCTGAGCGAGGGCTGCACCCCCTACGA CATCTGAGAATTCGCGGCCGC-3' AA Seq. MGNYVHLPLSPRTLNAWVKLIEEKKFGAEVVPGFQALSEGCTPYDI 5. SIVmac239Gag(178-258) DNA Seq. 5'- GTCGACAGGAGCCACCATGAGCGAGGGCTGCACCCCCTACGACATCAACC AGATGCTGAACTGCGTGGGCGACCACCAGGCCGCCATGCAGATCATCCGG GACATCATCAACGAGGAGGCCGCCGACTGGGACCTGCAGCACCCCCAGCC CGCCCCCCAGCAAGGCCAGCTGCGGGAGCCCAGCGGCAGCGACATCGCCG GCACCACCAGCAGCGTGGACGAGCAGATCCAGTGGATGTACCGGCAGCAG AACCCCATCCCCTGAGAATTCGCGGCCGC-3' AA Seq. MSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAADWDLQHPQPAPQQG QLREPSGSDIAGTTSSVDEQIQWMYRQQNPIP 6. SIVmac239Gag(250-415) DNA Seq. 5'- GTCGACAGGAGCCACCATGTACCGGCAGCAGAACCCCATCCCCGTGGGCA ACATCTACCGGCGGTGGATCCAGCTGGGCCTGCAGAAATGCGTGCGGATG TACAACCCCACCAACATCCTGGACGTGAAGCAGGGCCCCAAGGAGCCCTT CCAGAGCTACGTGGACCGGTTCTACAAGAGCCTGCGGGCCGAGCAGACCG ACGCCGCCGTGAAGAACTGGATGACCCAGACCCTGCTGATCCAGAACGCC AACCCCGACTGCAAGCTGGTGCTGAAGGGCCTGGGCGTGAACCCCACCCT GGAGGAGATGCTGACCGCCTGCCAGGGCGTGGGCGGCCCCGGCCAGAAGG CCCGGCTGATGGCCGAGGCCCTGAAGGAGGCCCTGGCCCCCGTGCCCATC CCCTTCGCCGCCGCCCAGCAGCGGGGCCCCCGGAAGCCCATCAAGTGCTG GAACTGCGGCAAGGAGGGCCACAGCGCCCGGCAGTGCCGGGCCCCCCGGC GGCAGGGCTGCTGGTGAGAGAATTCGCGGCCGC-3' AA Seq. MYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTNILDVKQGPKEPFQSYVD RFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGVNPTLEEMLT ACQGVGGPGQKARLMAEALKEALAPVPIPFAAAQQRGPRKPIKCWNCGKE GHSARQCRAPRRQGCW 7. SIVmac239Vif(1-110) DNA Seq. 5'- GTCGACAGGAGCCACCATGGAGGAGGAGAAGCGGTGGATCGCCGTGCCCA CCTGGCGGATCCCCGAGCGGCTGGAGCGGTGGCACAGCCTGATCAAGTAC CTGAAGTACAAGACCAAGGACCTGCAGAAGGTGTGCTACGTGCCCCACTT CAAAGTGGGCTGGGCCTGGTGGACCTGCAGCCGGGTGATCTTCCCCCTGC AAGAGGGCAGCCACCTGGAGGTGCAGGGCTACTGGCACCTGACCCCCGAG AAGGGCTGGCTGAGCACCTACGCCGTGCGGATCACCTGGTACAGCAAGAA CTTCTGGACCGACGTGACCCCCAACTACGCCGACATCCTGCTGCACTGAG AATTCGCGGCCGC-3' AA Seq. MEEEKRWIAVPTWRIPERLERWHSLIKYLKYKTKDLQKVCYVPHFKVGWA WWTCSRVIFPLQEGSHLEVQGYWHLTPEKGWLSTYAVRITWYSKNFWTDV TPNYADILLH 8. SIVmac239Vif(102-214) DNA Seq. 5'- GTCGACAGGAGCCACCATGCCCAACTACGCCGACATCCTGCTGCACAGCA CCTACTTCCCCTGCTTCACCGCCGGCGAGGTCCGGCGGGCCATCCGGGGC GAGCAGCTGCTGAGCTGCTGCCGGTTCCCCCGGGCCCACAAGTACCAGGT GCCCAGCCTGCAGTACCTGGCCCTGAAGGTGGTGAGCGACGTGCGGAGCC AGGGCGAGAACCCCACCTGGAAGCAGTGGCGGCGGGACAACCGGCGGGGC CTGCGGATGGCCAAGCAGAACAGCCGGGGCGACAAGCAGCGGGGCGGCAA GCCTCCCACCAAGGGCGCCAACTTCCCCGGCCTGGCCAAGGTGCTGGGCA TCCTGGCCTGAGAATTCGCGGCCGC-3' AA Seq. MPNYADILLHSTYFPCFTAGEVRRAIRGEQLLSCCRFPRAHKYQVPSLQY LALKVVSDVRSQGENPTWKQWRRDNRRGLRMAKQNSRGDKQRGGKPPTKG ANFPGLAKVLGILA 9. SIVmac239Nef(45-210) DNA Seq. 5'- GTCGACAGGAGCCACCATGGGCCTGGACAAGGGCCTGAGCAGCCTGAGCT GCGAGGGCCAGAAGTACAACCAGGGCCAGTACATGAACACCCCCTGGCGG AACCCCGCCGAGGAGCGGGAGAAGCTGGCCTACCGGAAGCAGAACATGGA CGACATCGACGAGGAGGACGACGACCTGGTGGGCGTGAGCGTGCGGCCCA AGGTGCCCCTGCGGACCATGAGCTACAAGCTGGCCATCGACATGAGCCAC TTCATCAAGGAGAAGGGCGGCCTGGAGGGCATCTACTACAGCGCCCGGCG GCACCGGATCCTGGACATCTACCTGGAGAAGGAGGAGGGCATCATCCCCG ACTGGCAGGACTACACCAGCGGCCCCGGCATCCGGTACCCCAAGACCTTC GGCTGGCTGTGGAAGCTGGTGCCCGTGAACGTGAGCGACGAGGCCCAGGA GGACGAGGAGCACTACCTGATGCACCCCGCCCAGACCAGCCAGTGGGACG ACCCCTGGGGCGAGGTGTGAGAATTCGCGGCCGC-3' AA Seq. MGLDKGLSSLSCEGQKYNQGQYMNTPWRNPAEEREKLAYRKQNMDDIDEE DDDLVGVSVRPKVPLRTMSYKLAIDMSHFIKEKGGLEGIYYSARRHRILD IYLEKEEGIIPDWQDYTSGPGIRYPKTFGWLWKLVPVNVSDEAQEDEEHY LMHPAQTSQWDDPWGEV

[0027] The viral vectors, recombinant bacteria, and nucleic acids used in the compounds and vaccines disclosed herein may encode one or more proteins of HIV or SIV or portions of one or more proteins of HIV or SIV. In some embodiments, nucleic acids may encode an epitope (for example, a T-cell epitope) which is not part of a known HIV or SIV functional protein or which is encoded by a HIV or SIV alternative open reading frame (i.e., a "cryptic" epitope). (See, for example, Maness et al., J. Exp. Med. (2007), 204:2505-12; and Cardinau et al., J. Exp. Med. (2004), 199:1053-1063, which are all incorporated by reference herein in their entireties). A cryptic epitope may be located in an open reading frame (ORF) whose translation is not typically observed in the course of HIV or SIV viral replication. For example, Maness et al. discloses a cryptic epitope in SIVmac239 called "cRW9", which is located in the +2 reading frame relative to the ORF encoding the envelope protein. This cryptic epitope is located in the same ORF that encodes exon 1 of the Rev protein, but it is at the 3' side ("downstream") of the donor processing site ("donor splicing site") and so its translation would not be expected under "normal" biological conditions.

[0028] Any of the conventional vectors used for expression in eukaryotic cells may be used to directly immunize an individual with nucleic acid. Expression vectors containing regulatory elements of eukaryotic viruses may be used in eukaryotic expression vectors (for example, vectors containing SV40, CMV, or retroviral enhancers or promoters). Exemplary vectors include vectors expressing proteins under the direction of such promoters as SV40 early promoter, SV40 late promoter, metallothionein promoter, human cytomegalovirus promoter, murine mammary tumor virus promoter, and Rous sarcoma virus promoter. Industrial amounts for therapeutic and prophylactic use of plasmid DNA may be yielded, for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate a culture medium, and cultured until saturation in shake flasks or in a bioreactor according to widely known techniques. Plasmid DNA may be purified by standard bioseparation technologies such as solid phase ion exchange resins. If required, the DNA may be isolated from the open-circular and linear forms using gel electrophoresis or other methods.

[0029] The purified plasmid DNA may be prepared for injection using a wide range of formulations, such as, for example, lyophilized DNA which may be reconstituted in phosphate-buffered sterile saline (PBS) solution. The purified DNA may be introduced into an individual through any appropriate method; for example, by intramuscular (IM) or intradermal (ID) administration.

[0030] In order to maximize the immunotherapy effects of DNA vaccines, alternative methods may be desirable to formulate purified plasmid DNA. A variety of methods has been described, and new techniques may become available. For example, cationic lipids may be used in the formulation of the pharmaceutical composition disclosed herein (for example, as described in WO 93/24640; U.S. Pat. No. 5,279,833; and WO 91/06309). Protective, interactive, noncondensing compounds (PINC) may be used in the formulation of the pharmaceutical composition disclosed herein (for example, glycolipids, fusogenic liposomes, and peptides).

[0031] The term "vector" refers to a few means by which nucleic acid may be introduced into a host organism or tissue. There are several types of vectors, including viruses, plasmids, bacteriophages, cosmids, and bacteria. As used herein, a "recombinant virus" or "viral vector" relates to the recombinant viral nucleic acid which has been genetically modified to express a heterologous polypeptide (for example, a HIV polypeptide or a fragment thereof). The recombinant viral nucleic acid typically includes cis-acting elements for the expression of the heterologous polypeptide. Typically, the recombinant viral nucleic acid can be packaged into a virus which is able to infect a host cell. For example, the recombinant viral nucleic acid may include cis-acting elements for packaging. Generally, the viral vector is not able to replicate or is attenuated. An "attenuated recombinant virus" refers to a virus which has been genetically modified by modern molecular biological methods, making it less virulent than the wild type, usually by mutation or deletion of specific genes. For example, the recombinant viral nucleic acid may not have an essential gene for an efficient production or for the production of the infectious virus.

[0032] The recombinant viral nucleic acid may function as a vector for an immunogenic retroviral protein in view of the fact that the recombinant viral nucleic acid comprises exogenous nucleic acid. The recombinant virus may be introduced in a vaccinated human by standard methods for vaccination using live vaccines. A live vaccine as contemplated herein may be administered using, for example, from about 10.sup.4 to 10.sup.8 units of viral particles/dose, or 10.sup.6 to 10.sup.9 pfu/dose. The actual dosages of this vaccine may be readily determined by any expert in the vaccine field.

[0033] Numerous virus species may be used as recombinant virus vectors for the pharmaceutical composition disclosed herein. Preferred recombinant virus for a viral vaccine is the yellow fever vaccine virus (for example, 17-D or similar).

[0034] In some embodiments, attenuated recombinant bacteria or mycobacteria may be used as vectors in the pharmaceutical compositions and vaccines disclosed herein (for example, Bacillus, Shigella, Salmonella, Listeria or Yersinia, attenuated recombinant bacteria). Recombinant bacterial vaccine vectors are described by Daudel et al., "Use of attenuated bacteria as delivery vectors for DNA vaccines", Expert Review of Vaccines, Volume 6, Number 1, February, 2007, pp. 97-110(14); Shata et al., "Recent advances with recombinant bacterial vaccine vectors", Molec. Med. Today (2000), Volume 6, Number 2, Feb. 1, 2000, pages 66-71; Clare & Dougan, "Live Recombinant Bacterial Vaccines", Novel Vaccination Strategies, Apr. 16, 2004 (Editor Stefan H. E. Kaufman); Gentaschev et al., "Recombinant Attenuated Bacteria for the Delivery of Subunit Vaccines", Vaccine, Volume 19, Numbers 17-19, Mar. 21, 2001, pages 2621-2628; Garmory et al., "The Use of live attenuated bacteria as a delivery system for heterologous antigens", J. Drug Target. 2003; 11 (8-10): 471-9; U.S. Pat. No. 6,383,496; and U.S. Pat. No. 6,923,958 (which are all incorporated by reference herein in their entireties).

[0035] In some embodiments, the recombinant bacterium or mycobacterium is the recombinant Bacillus Calmette-Guerin (or Bacillus Calmette-Guerin, BCG). The BCG is an attenuated strain of the live bovine tuberculosis bacillus, Mycobacterium bovis, which lost its virulence in humans by being specially cultured in an artificial medium over years. The use of a recombinant BCG (rBCG) expressing the codon-optimized HIV-1 Gag for mice immunization in a "prime-boost" regimen has been described. Promkhatkaaew D. et al., "Prime-boost immunization of codon optimized HIV-1 CRFO1 AE Gag in BCG with recombinant vaccinia virus elicits MHC class I and II immune responses in mice", Immunol Invest. 2009; 38(8):762-79.

[0036] The compositions included in the vaccine regimen as contemplated herein may be coadministered or administered sequentially with another immunological antigenic vaccine or in therapeutic compositions, including an adjuvant, a biological or chemical agent given in combination with or genetically fused to an antigen to increase the antigen immunogenicity. Additional therapeutic agents may include but are not limited to CD40 or interleukin-2 (IL-2) ligand in a sufficient amount to further potentiate CD8+ T cell, CD4+ T cell, and antibody responses. These other compositions may also include purified antigens of the immunodeficiency virus or the expression of such antigens by a second recombinant vector system which can give rise to additional therapeutic compositions.

[0037] The pharmaceutical composition disclosed herein may be delivered by various routes. The typical delivery routes include parenteral administration (for example, intradermal, intramuscular, or subcutaneous delivery). Other routes include oral, intranasal, intravaginal, and intrarectal administration. The pharmaceutical composition may be formulated for intranasal or pulmonary delivery. The pharmaceutical composition formulations may include liquid formulations (for example, for oral, nasal, anal, or vaginal administration, among others, including suspensions, syrups, or elixirs) and preparations for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (for example, injectable administration) such as sterile suspensions or emulsions.

[0038] As used herein, the term "adjuvant" refers to a compound or mixture which increases the immune response to an antigen. An adjuvant may serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator which nonspecifically amplifies the immune response. Examples of adjuvants which may be employed include the adjuvant MPL-TDM (Monophosphoryl lipid A/synthetic trehalose dicorynomycolate; for example, available from GSK Biologicals). Another appropriate adjuvant is the AS021/AS02 immunostimulatory adjuvant (GSK). These immunostimulatory adjuvants are formulated to promote an intense T-cell response and include QS-21, a saponin from Quillay saponaria, the TL4 ligand, a monophosphoryl lipid A, together in a lipid or liposomal carrier. Other adjuvants include but are not limited to nonionic block copolymer adjuvants (for example, CRL1005), aluminum phosphates (for example, A1PO4), R-848 (a Th1-like adjuvant), imiquimod, PAM3CYS, poly (I:C), loxoribine, potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), antigens derived from cholera toxin (for example, CTA1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions in water (for example, MF59 available from Novartis Vaccines or Montanide ISA 720), keyhole-limpet hemocyanins, and dinitrophenol.

[0039] The pharmaceutical compositions disclosed herein may also or additionally comprise at least one antiviral chemotherapy compound. Nonlimiting examples may be selected from at least one compound of the group consisting of gamma globulin, amantadine, guanidine, benzimidazole hydroxide, interferons, interleukin-16, thiosemicarbazones, methisazones, rifampicin, ribavirin, pyrimidine analogs (for example, AZT and/or 3TC), purine analogs, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, protease inhibitors (for example, saquinavir; indinavir; ritonavir; AG 1343; and VX-2/78), chemokines, such as RANTES, MIP1x or MIP1b, or ganciclovir.

[0040] As used herein, a "prime-boost vaccination regimen" refers to a regimen in which a first composition is administered to an individual one or more times (for example, two or three times with about 2, 3, or 4 weeks between administrations), and, after a certain period of time (for example, about two weeks, about four weeks, about two months, about three months, about four months, about five months, about six months, or more), a second composition is administered to the individual, which may be equal to or different from the first composition. The second composition may also be administered more than once, with at least 2, 3, or 4 weeks between administrations. In some embodiments, the individual is given the first composition, which comprises the recombinant yellow fever virus 17D expressing one or more HIV or SIV polypeptides, or their fragments, or rBCG expressing one or more HIV or SIV polypeptides, or their fragments. Subsequently, the individual receives the second composition, which comprises the recombinant yellow fever virus 17D expressing one or more HIV or SIV polypeptides, or their fragments, DNA encoding one or more HIV or SIV polypeptides, or their fragments, or rBCG expressing one or more HIV or SIV polypeptides or their fragments. The first and the second compositions may be the same or different.

[0041] The pharmaceutical compositions disclosed herein may be delivered to individuals at risk of developing HIV or SIV infection or to individuals who are already infected with HIV or SIV. To evaluate the efficacy of the vaccine, the immune response may be assessed by measuring the induction of CD8+ and antibody responses for particular epitopes. CD8+ T-cell responses may be measured, for example, by using cultured or fresh PBMC tetramer staining, ELISPOT assays, or functional cytotoxicity assays, which are well known among experts, and are described herein. Antibody responses may be measured by widely known assays such as ELISA. Determinations of viral titers or loads and CD4+ T cells after immunization may be performed in individuals who have already been infected. For example, individuals may present a decline in the viral load and an increase in the CD4+cell count after immunization (for example, at least a twofold, threefold, or fourfold increase in the CD4+cell count in relation to the count obtained prior to immunization).

[0042] The invention will now be described in detail regarding certain embodiments of the invention.

[0043] In some embodiments of the recombinant viruses contemplated herein, the virus is a yellow fever vaccinal virus engineered to express a heterologous polypeptide. The heterologous polypeptide may include a HIV polypeptide (HIV-1 or HIV-2), a SIV polypeptide, or a fragment of them (for example, a fragment consisting of at least 8, 9, 10, 20, 30, 40, 50, 100, or 200 contiguous amino acids of a HIV Gag polypeptide or a SIV Gag polypeptide). In other embodiments, the heterologous polypeptide is a polypeptide with at least 90%, 95%, 96%, 97%, 98%, or 99% of sequence identity with a HIV polypeptide, a SIV polypeptide, or a fragment of them (for example, a fragment consisting of at least 8, 9, 10, 20, 30, 40, 50, 100, or 200 contiguous amino acids of a HIV Gag polypeptide or a SIV Gag polypeptide). Sequences for HIV-1 strains and several genes of HIV-1 strains (including the gene for Gag polypeptide) are publicly available in GenBank; for example, the sequences available under accession numbers: NC.sub.--001802; EU293448, EU293447, EU293446, EU293445, EU293444, FJ195091, FJ195090, FJ195089, FJ195088, FJ195087, FJ195086, EU884501, EU786681, EU786680, EU786679, EU786678, EU786677, EU786676, EU786675, EU786674, EU786673, EU786672, EU786671, EU786670, EU697909, EU697908, EU697907, EU697906, EU697905, EU697904, U71182, EU69324, EU861977, FJ213783, FJ213782, FJ213781, FJ213780, AB428562, AB428561, AB428560, AB428559, AB428558, AB428557, AB428556, AB428555, AB428554, AB428553, AB428552, AB428551, DQ295195, DQ295196, DQ295194, DQ295193, DQ295192, EU446022, EU735540, EU735539, EU735538, EU735537, EU735536, EU735535, EU110097, EU110096, EU110095, EU110094, EU110093, EU110092, EU110091, EU110090, EU110089, EU110088, EU110087, EU110086, EU110085, AF193277, AF193276, AF049337, EU541617, EU220698, EF469243, DQ912823, DQ912822, EU000516, EU000515, EU000514, EU000513, EU000512, EU000511, EU000510, EU000509, EU000508, EU000507, and EU884500; whose submissions to GenBank are incorporated by reference herein in their entireties. Primary and field isolates of HIV are available from the National Institute of Allergy and Infectious Diseases (NIAID), which has contracted the services of Quality Biological (Gaithersburg, Md.) to make these strains available. Strains are also available from the World Health Organization (WHO), Geneva, Switzerland. In a preferred embodiment, the selected polypeptide has a consensus sequence as determined by the comparison of three or more HIV strains. As disclosed herein, the polynucleotides encoding a HIV polypeptide or a SIV polypeptide may be "optimized" for expression in a human cellular environment or simian cellular environment, respectively. Thus, the use of polynucleotides specifically including HIV genes that are codon-optimized for expression in a human cellular environment is contemplated herein. Optimized synthetic HIV gag genes are described in U.S. Pat. No. 6,696,291.

[0044] The recombinant yellow fever viruses contemplated herein are prepared through the insertion of the sequence encoding the heterologous polypeptide into the genome of the yellow fever virus or vector which can replicate the yellow fever virus. Adequate yellow fever viruses include attenuated strains such as YF17D. Adequate insertion sites for the sequence encoding the heterologous polypeptide include sites between the sequences encoding viral proteins E and NS1. The sequence encoding the heterologous polypeptide may be codon-optimized based on the use of the codon for the yellow fever virus genome.

[0045] In another embodiment of the recombinant viruses described herein, the virus is a genetically modified vaccinal YF 17D virus which expresses amino acids 45-269 of SIVmac239Gag (rYF17D/SIVGag45-269), and is prepared through the insertion of a codon-optimized sequence based on YF genome between the genes which encode viral proteins E and NS1.

[0046] In one example, the antigen used for expression by the yellow fever 17D virus was a fragment of a Gag protein comprising amino acid residues 45 to 269. Gag is a precursory protein that, on being processed by the viral protease, generates the internal structural proteins of the mature virion. Gag protein induces a strong protective immune response mediated by CD8+ T cells in rhesus monkeys and, on a smaller scale, in humans. This immune response is very important since it may provide protection against viral infection or reduce the viral load and delay the onset of disease.

[0047] In some embodiments, the following criteria may be used to select a Gag fragment to be cloned and expressed in the E/NS1 region of 17D virus genome. First, the presence of CD8+ T-cell epitopes in the Gag polypeptide may be taken into consideration based on immunological studies in rhesus monkeys. Furthermore, preferably, the insertion of the Gag polypeptide should not disturb the translocation of the YF precursory protein into the endoplasmic reticulum, as well as its processing by viral and cell proteases, if these steps occur during virus morphogenesis and assembly. Thus, preferably, the Gag polypeptide fragment should not be hydrophobic or contain a transmembrane motif, which might produce a rupture or significantly destabilize the correct topology of the membrane and precursory protein processing. Therefore, the selected Gag polypeptide fragment is hydrophilic and does not include apparent transmembrane domains. Besides, a very flexible motif, KESSIG, is fused to the C-terminal of the Gag sequence. This sort of motif is present in all flaviviruses and proteins binding domain III and the anchorage region ("stem anchor") of E protein. It is a very flexible motif and was used here as a separating sequence to connect and move away the Gag fragment and alpha helices of the anchorage domain ("stem anchor") of truncated dengue 4 virus E protein in the recombinant cassette.

[0048] Moreover, the virus vaccines presently disclosed differ from the virus vaccines of the state of the art by using an attenuated vector structure and emphasizing the generation of cell immune responses for specific viral epitopes in certain proteins. The strategy for vector virus replication is maximizing the production of antigens in an appropriate cell location to induce an appropriate response in terms of specificity and magnitude.

[0049] Vaccination with a single dose of attenuated yellow fever virus 17D causes a limited viral infection and promotes the generation of neutralizing antibodies as well as T-cell responses which confer protective immunity to more than 95% of vaccinees, providing immunity for over 30 years. The yellow fever 17D vaccine is regarded as one of the most successful vaccines available. Vaccine production concerns a well-established process and, since 1937, over 540 million doses have been used in humans with a minimal incidence of severe side effects [9,10].

[0050] The current methods are based on the methodology that was previously developed by two of the present inventors. The methodology is described in WO07051267, which is incorporated by reference herein.

[0051] The method for the production of recombinant viruses with nucleotide sequences totally or partially encoding the heterologous proteins, as described in WO07051267, may comprise the following steps:

[0052] (a) modification of the heterologous nucleotide sequences in such a way that, when they are cloned in the vector virus, they present, in the 5' region, nucleotides of the 5' end of the vector virus NS1 gene or functionally equivalent sequences, and, in its 3' end, the totality or part of the anchorage genome region ("stem anchor") of E protein of the vector virus or functionally equivalent sequences, which do not compromise the structure and replication of said vector virus;

[0053] (b) insertion of the modified heterologous sequences as described in (a) into the intergenic region between the genes encoding E structural protein and NS1 nonstructural protein of the vector virus;

[0054] (c) regeneration of the nonpathogenic recombinant virus, having the heterologous sequences stably integrated into the viral genome at the insertion point described in (b), and expression of the heterologous sequence in such a way that the corresponding protein induces an appropriate immune response.

[0055] Thus, the inventors found that the strategy for E/NS1 insertion may be applied to generate recombinant vaccines against lentiviruses, particularly the HIV (Human Immunodeficiency Virus).

In one embodiment of a recombinant virus as contemplated herein, an attenuated recombinant virus is a yellow fever vaccinal virus 17D expressing a sequence of SIVmac239 Gag sequences which is used as a viral vector to originate SIV-specific CD8+ T-cell responses in rhesus monkeys. The model of Asian rhesus monkey was employed to validate the expression of HIV antigens in the yellow fever 17D virus vector because the SIV-infected rhesus monkey model reproduces, in many aspects, the immunodeficiency that occurs in HIV-infected humans. First, several SIV strains are closely related to HIV-1 strains and infect CD4+ T cells, giving rise to a decline in their counts over time. SIV infection causes a disease that is similar to AIDS in most of the monkeys infected for one year after inoculation. Moreover, many genes of the monkeys which encode key proteins in the immune system are similar to those of humans. Equivalents of the genes for HLA class, class II [sic] and T-cell receptor (TCR) are found in the monkeys. Orthologs of HLA-A, -B, -E and -F locations were isolated in rhesus monkeys. In both infections, CD8+ T cells select the major part of the sequence variation outside the envelope protein and the natural control of viral replication is associated with certain MHC-I alleles [11,12].

[0056] Consequently, as disclosed herein, the vaccination of rhesus monkeys with different formulations containing recombinant yellow fever virus was assessed so as to determine the potential use of 17D vector in the development of new vaccines against HIV. The recombinant yellow fever virus obtained expresses a SIVmac239 Gag fragment encompassing amino acids 45 to 269. Gag is a precursory protein and, in the course of viral replication, it undergoes proteolytic splitting by the viral protease, producing the internal structural proteins of the mature virion. It was determined that multiple Gag-specific responses are important in the control of SIV replication. This may be due to infected cells which present Gag-derived CD8+ T-cell epitopes right after the viral entry. Gag-specific CD8+ T cells recognize infected cells in up to 2 hours after the infection. Six hours after the infection, Gag-specific CD8+ T cells eliminate the infected cells, both before and after the integration of proviral DNA and viral protein synthesis. These findings demonstrate that Gag-specific CD8+ T cells do not require productive cell infection or even protein synthesis "again" to recognize the infected cells and eliminate them. This suggests that Gag-specific CD8+ T cells may be much more important than previously imagined.

[0057] In order to reach the goals disclosed herein, the following sequences were used:

TABLE-US-00008 SEQ. ID. NO.: 1 ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005## FYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGVNPTLEEMLTA CQGVGGPGQKARLMAEALKEALAPVPIPFAAAQQRGPRKTIKCWNCGKEG HSARQCRAPRRQGCWKCGKMDHVMAKCPDRQAGFLGLGPWGKKPRNFPMA QVHQGLMPTAPPEDPAVDLLKNYMQLGKQQREKQRESRXKPYKEVTEDLL HLNSLFGGDQ.

(TO BE CONFIRMED, but in several seqs. of the GENBANK submitted by David's group, the seqs. have 510 amino acids--the final Q residue is missing).

[0058] SEQ. ID. NO.: 2 is a sequence of amino acids of the recombinant protein Gag 45-269, cloned and expressed in the E/NS1 intergenic region by the yellow fever 17D virus. Gag45-269 fragment is shaded in gray.

TABLE-US-00009 SEQ. ID. NO.: 2 ##STR00006## ##STR00007## ##STR00008## ##STR00009## VFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLF LGFTVGA

Wherein the other sequences present in SEQ. ID. NO.: 2 are sequences of the yellow fever virus and correspond to:

TABLE-US-00010 SEQ. ID. NO.: 3 ##STR00010##

[0059] SEQ. ID. NO.: 3 is a variation of 10 amino acid residues of the N-terminal sequence of NS1 protein of the yellow fever virus (DQGAINFGK); YF genome position from 2453 to 2482)[sic] in which an amino acid replacement was introduced (from cysteine to serine, shaded in gray).

TABLE-US-00011 SEQ. ID. NO.: 4 KESSIG

[0060] SEQ. ID. NO.: 4 is the sequence of amino acids corresponding both to the sequence of nucleotides of the position from 2145 to 2164 of the yellow fever virus genome and the motif comprising the amino acid residues 391 to 397 of E protein of the yellow fever virus.

TABLE-US-00012 SEQ. ID. NO.: .5 TSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTC IAVGGITLFLGFTVGA

[0061] SEQ. ID. NO.: 5 is a variation of the last-terminal [sic] amino acid residues of E protein of dengue virus serotype 4 (dengue virus serotype 4 genome position from 2226 to 2423; GenBank AF326825). SEQ. ID. NO.: 5 includes a replacement relative to the original sequence where the amino acid terminal sequence VQA is changed to VGA.

[0062] SEQ. ID. NO.: 6 is a nucleotide sequence (924 nucleotides) of the cassette of recombinant Gag45-269, cloned in the E/NS1 intergenic region of the yellow fever 17D virus. Gag 45-269 fragment is shaded in gray.

TABLE-US-00013 SEQ. ID. NO.: 6 ##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ACCAGGTTTTTGGAAGTGTGTATACAACCATGTTTGGAGGAGTCTCATGG ATGATTAGAATCCTAATTGGGTTCTTAGTGTTGTGGATTGGCACGAACTC CAGGAACACTTCAATGGCTATGACGTGCATAGCTGTTGGAGGAATCACTC TGTTTCTGGGCTTCACAGTTGGCGCC 3'

[0063] SEQ. ID. NO.: 7 is a sequence of 11, 785 [sic] nucleotides of the recombinant virus genome taking cassette Gag 45-269, called rYF17D/SIVGag45-269.

TABLE-US-00014 SEQ. ID. NO.: 7 5' AGTAAATCCTGTGTGCTAATTGAGGTGCATTGGTCTGCAAATCGAGTTGC TAGGCAATAAACACATTTGGATTAATTTTAATCGTTCGTTGAGCGATTAG CAGAGAACTGACCAGAACATGTCTGGTCGTAAAGCTCAGGGAAAAACCCT GGGCGTCAATATGGTACGACGAGGAGTTCGCTCCTTGTCAAACAAAATAA AACAAAAAACAAAACAAATTGGAAACAGACCTGGACCTTCAAGAGGTGTT CAAGGATTTATCTTTTTCTTTTTGTTCAACATTTTGACTGGAAAAAAGAT CACAGCCCACCTAAAGAGGTTGTGGAAAATGCTGGACCCAAGACAAGGCT TGGCTGTTCTAAGGAAAGTCAAGAGAGTGGTGGCCAGTTTGATGAGAGGA TTGTCCTCAAGGAAACGCCGTTCCCATGATGTTCTGACTGTGCAATTCCT AATTTTGGGAATGCTGTTGATGACCGGTGGAGTGACCTTGGTGCGGAAAA ACAGATGGTTGCTCCTAAATGTGACATCTGAGGACCTCGGGAAAACATTC TCTGTGGGCACAGGCAACTGCACAACAAACATTTTGGAAGCCAAGTACTG GTGCCCAGACTCAATGGAATACAACTGTCCCAATCTCAGTCCAAGAGAGG AGCCAGATGACATTGATTGCTGGTGCTATGGGGTGGAAAACGTTAGAGTC GCATATGGTAAGTGTGACTCAGCAGGCAGGTCTAGGAGGTCAAGAAGGGC CATTGACTTGCCTACGCATGAAAACCATGGTTTGAAGACCCGGCAAGAAA AATGGATGACTGGAAGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGA TGGTTCGTGAGGAACCCCTTTTTTGCAGTGACGGCTCTGACCATTGCCTA CCTTGTGGGAAGCAACATGACGCAACGAGTCGTGATTGCCCTACTGGTCT TGGCTGTTGGTCCGGCCTACTCAGCTCACTGCATTGGAATTACTGACAGG GATTTCATTGAGGGGGTGCATGGAGGAACTTGGGTTTCAGCTACCCTGGA GCAAGACAAGTGTGTCACTGTTATGGCCCCTGACAAGCCTTCATTGGACA TCTCACTAGAGACAGTAGCCATTGATAGACCTGCTGAGGCGAGGAAAGTG TGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCCCAG CACTGGAGAGGCCCACCTAGCTGAAGAGAACGAAGGGGACAATGCGTGCA AGCGCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGG AAAGGGAGCATTGTGGCATGCGCCAAATTCACTTGTGCCAAATCCATGAG TTTGTTTGAGGTTGATCAGACCAAAATTCAGTATGTCATCAGAGCACAAT TGCATGTAGGGGCCAAGCAGGAAAATTGGAATACCAGCATTAAGACTCTC AAGTTTGATGCCCTGTCAGGCTCCCAGGAAGTCGAGTTCATTGGGTATGG AAAAGCTACACTGGAATGCCAGGTGCAAACTGCGGTGGACTTTGGTAACA GTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGGACAGACAGTGG GCCCAGGACTTGACCCTGCCATGGCAGAGTGGAAGTGGCGGGGTGTGGAG AGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCA GAGTACTGGCCCTGGGAAACCAGGAAGGCTCCTTGAAAACAGCTCTTACT GGCGCAATGAGGGTTACAAAGGACACAAATGACAACAACCTTTACAAACT ACATGGTGGACATGTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCA AGGGGACATCCTACAAAATATGCACTGACAAAATGTTTTTTGTCAAGAAC CCAACTGACACTGGCCATGGCACTGTTGTGATGCAGGTGAAAGTGTCAAA AGGAACCCCCTGCAGGATTCCAGTGATAGTAGCTGATGATCTTACAGCGG CAATCAATAAAGGCATTTTGGTTACAGTTAACCCCATCGCCTCAACCAAT GATGATGAAGTGCTGATTGAGGTGAACCCACCTTTTGGAGACAGCTACAT TATCGTTGGGAGAGGAGATTCACGTCTCACTTACCAGTGGCACAAAGAGG GAAGCTCAATAGGAAAGTTGTTCACTCAGACCATGAAAGGCGTGGAACGC CTGGCCGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTT CTTCACTTCGGTTGGGAAAGGAATTCATACGGTGTTTGGCTCTGCCTTTC AGGGGCTATTTGGCGGCTTGAACTGGATAACAAAGGTCATCATGGGGGCG GTACTCATATGGGTTGGCATCAACACAAGAAACATGACAATGTCCATGAG CATGATCTTGGTAGGAGTGATCATGATGTTTTTGTCTCTAGGAGTTGGGG ##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## GGCAAGAGAGAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCT GATGACTGGCTGAACAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTT GCATCAATAGTGAAAGCCTCTTTCGAAGAAGGGAAGTGTGGCCTAAATTCA GTTGACTCCCTTGAGCATGAGATGTGGAGAAGCAGGGCAGATGAGATTAAT ACCATTTTTGAGGAAAACGAGGTGGACATTTCTGTTGTCGTGCAGGATCCA AAGAATGTTTACCAGAGAGGAACTCATCCATTTTCCAGAATTCGGGATGGT CTGCAGTATGGTTGGAAGACTTGGGGTAAGAACCTTGTGTTCTCCCCAGGG AGGAAGAATGGAAGCTTCATCATAGATGGAAAGTCCAGGAAAGAATGCCCG TTTTCAAACCGGGTCTGGAATTCTTTCCAGATAGAGGAGTTTGGGACGGGA GTGTTCACCACACGCGTGTACATGGACGCAGTCTTTGAATACACCATAGAC TGCGATGGATCTATCTTGGGTGCAGCGGTGAACGGAAAAAAGAGTGCCCAT GGCTCTCCAACATTTTGGATGGGAAGTCATGAAGTAAATGGGACATGGATG ATCCACACCTTGGAGGCATTAGATTACAAGGAGTGTGAGTGGCCACTGACA CATACGATTGGAACATCAGTTGAAGAGAGTGAAATGTTCATGCCGAGATCA ATCGGAGGCCCAGTTAGCTCTCACAATCATATCCCTGGATACAAGGTTCAG ACGAACGGACCTTGGATGCAGGTACCACTAGAAGTGAAGAGAGAAGCTTGC CCAGGGACTAGCGTGATCATTGATGGCAACTGTGATGGACGGGGAAAATCA ACCAGATCCACCACGGATAGCGGGAAAGTTATTCCTGAATGGTGTTGCCGC TCCTGCACAATGCCGCCTGTGAGCTTCCATGGTAGTGATGGGTGTTGGTAT CCCATGGAAATTAGGCCAAGGAAAACGCATGAAAGCCATCTGGTGCGCTCC TGGGTTACAGCTGGAGAAATACATGCTGTCCCTTTTGGTTTGGTGAGCATG ATGATAGCAATGGAAGTGGTCCTAAGGAAAAGACAGGGACCAAAGCAAATG TTGGTTGGAGGAGTAGTGCTCTTGGGAGCAATGCTGGTCGGGCAAGTAACT CTCCTTGATTTGCTGAAACTCACAGTGGCTGTGGGATTGCATTTCCATGAG ATGAACAATGGAGGAGACGCCATGTATATGGCGTTGATTGCTGCCTTTTCA ATCAGACCAGGGCTGCTCATCGGCTTTGGGCTCAGGACCCTATGGAGCCCT CGGGAACGCCTTGTGCTGACCCTAGGAGCAGCCATGGTGGAGATTGCCTTG GGTGGCGTGATGGGCGGCCTGTGGAAGTATCTAAATGCAGTTTCTCTCTGC ATCCTGACAATAAATGCTGTTGCTTCTAGGAAAGCATCAAATACCATCTTG CCCCTCATGGCTCTGTTGACACCTGTCACTATGGCTGAGGTGAGACTTGCC GCAATGTTCTTTTGTGCCATGGTTATCATAGGGGTCCTTCACCAGAATTTC AAGGACACCTCCATGCAGAAGACTATACCTCTGGTGGCCCTCACACTCACA TCTTACCTGGGCTTGACACAACCTTTTTTGGGCCTGTGTGCATTTCTGGCA ACCCGCATATTTGGGCGAAGGAGTATCCCAGTGAATGAGGCACTCGCAGCA GCTGGTCTAGTGGGAGTGCTGGCAGGACTGGCTTTTCAGGAGATGGAGAAC TTCCTTGGTCCGATTGCAGTTGGAGGACTCCTGATGATGCTGGTTAGCGTG GCTGGGAGGGTGGATGGGCTAGAGCTCAAGAAGCTTGGTGAAGTTTCATGG GAAGAGGAGGCGGAGATCAGCGGGAGTTCCGCCCGCTATGATGTGGCACTC AGTGAACAAGGGGAGTTCAAGCTGCTTTCTGAAGAGAAAGTGCCATGGGAC CAGGTTGTGATGACCTCGCTGGCCTTGGTTGGGGCTGCCCTCCATCCATTT GCTCTTCTGCTGGTCCTTGCTGGGTGGCTGTTTCATGTCAGGGGAGCTAGG AGAAGTGGGGATGTCTTGTGGGATATTCCCACTCCTAAGATCATCGAGGAA TGTGAACATCTGGAGGATGGGATTTATGGCATATTCCAGTCAACCTTCTTG GGGGCCTCCCAGCGAGGAGTGGGAGTGGCACAGGGAGGGGTGTTCCACACA ATGTGGCATGTCACAAGAGGAGCTTTCCTTGTCAGGAATGGCAAGAAGTTG ATTCCATCTTGGGCTTCAGTAAAGGAAGACCTTGTCGCCTATGGTGGCTCA TGGAAGTTGGAAGGCAGATGGGATGGAGAGGAAGAGGTCCAGTTGATCGCG GCTGTTCCAGGAAAGAACGTGGTCAACGTCCAGACAAAACCGAGCTTGTTC AAAGTGAGGAATGGGGGAGAAATCGGGGCTGTCGCTCTTGACTATCCGAGT GGCACTTCAGGATCTCCTATTGTTAACAGGAACGGAGAGGTGATTGGGCTG TACGGCAATGGCATCCTTGTCGGTGACAACTCCTTCGTGTCCGCCATATCC CAGACTGAGGTGAAGGAAGAAGGAAAGGAGGAGCTCCAAGAGATCCCGACA ATGCTAAAGAAAGGAATGACAACTGTCCTTGATTTTCATCCTGGAGCTGGG AAGACAAGACGTTTCCTCCCACAGATCTTGGCCGAGTGCGCACGGAGACGC TTGCGCACTCTTGTGTTGGCCCCCACCAGGGTTGTTCTTTCTGAAATGAAG GAGGCTTTTCACGGCCTGGACGTGAAATTCCACACACAGGCTTTTTCCGCT CACGGCAGCGGGAGAGAAGTCATTGATGCCATGTGCCATGCCACCCTAACT TACAGGATGTTGGAACCAACTAGGGTTGTTAACTGGGAAGTGATCATTATG GATGAAGCCCATTTTTTGGATCCAGCTAGCATAGCCGCTAGAGGTTGGGCA GCGCACAGAGCTAGGGCAAATGAAAGTGCAACAATCTTGATGACAGCCACA CCGCCTGGGACTAGTGATGAATTTCCACATTCAAATGGTGAAATAGAAGAT GTTCAAACGGACATACCCAGTGAGCCCTGGAACACAGGGCATGACTGGATC CTGGCTGACAAAAGGCCCACGGCATGGTTCCTTCCATCCATCAGAGCTGCA AATGTCATGGCTGCCTCTTTGCGTAAGGCTGGAAAGAGTGTGGTGGTCCTG AACAGGAAAACCTTTGAGAGAGAATACCCCACGATAAAGCAGAAGAAACCT GACTTTATATTGGCCACTGACATAGCTGAAATGGGAGCCAACCTTTGCGTG GAGCGAGTGCTGGATTGCAGGACGGCTTTTAAGCCTGTGCTTGTGGATGAA

GGGAGGAAGGTGGCAATAAAAGGGCCACTTCGTATCTCCGCATCCTCTGCT GCTCAAAGGAGGGGGCGCATTGGGAGAAATCCCAACAGAGATGGAGACTCA TACTACTATTCTGAGCCTACAAGTGAAAATAATGCCCACCACGTCTGCTGG TTGGAGGCCTCAATGCTCTTGGACAACATGGAGGTGAGGGGTGGAATGGTC GCCCCACTCTATGGCGTTGAAGGAACTAAAACACCAGTTTCCCCTGGTGAA ATGAGACTGAGGGATGACCAGAGGAAAGTCTTCAGAGAACTAGTGAGGAAT TGTGACCTGCCCGTTTGGCTTTCGTGGCAAGTGGCCAAGGCTGGTTTGAAG ACGAATGATCGTAAGTGGTGTTTTGAAGGCCCTGAGGAACATGAGATCTTG AATGACAGCGGTGAAACAGTGAAGTGCAGGGCTCCTGGAGGAGCAAAGAAG CCTCTGCGCCCAAGGTGGTGTGATGAAAGGGTGTCATCTGACCAGAGTGCG CTGTCTGAATTTATTAAGTTTGCTGAAGGTAGGAGGGGAGCTGCTGAAGTG CTAGTTGTGCTGAGTGAACTCCCTGATTTCCTGGCTAAAAAAGGTGGAGAG GCAATGGATACCATCAGTGTGTTCCTCCACTCTGAGGAAGGCTCTAGGGCT TACCGCAATGCACTATCAATGATGCCTGAGGCAATGACAATAGTCATGCTG TTTATACTGGCTGGACTACTGACATCGGGAATGGTCATCTTTTTCATGTCT CCCAAAGGCATCAGTAGAATGTCTATGGCGATGGGCACAATGGCCGGCTGT GGATATCTCATGTTCCTTGGAGGCGTCAAACCCACTCACATCTCCTATGTC ATGCTCATATTCTTTGTCCTGATGGTGGTTGTGATCCCCGAGCCAGGGCAA CAAAGGTCCATCCAAGACAACCAAGTGGCATACCTCATTATTGGCATCCTG ACGCTGGTTTCAGCGGTGGCAGCCAACGAGCTAGGCATGCTGGAGAAAACC AAAGAGGACCTCTTTGGGAAGAAGAACTTAATTCCATCTAGTGCTTCACCC TGGAGTTGGCCGGATCTTGACCTGAAGCCAGGAGCTGCCTGGACAGTGTAC GTTGGCATTGTTACAATGCTCTCTCCAATGTTGCACCACTGGATCAAAGTC GAATATGGCAACCTGTCTCTGTCTGGAATAGCCCAGTCAGCCTCAGTCCTT TCTTTCATGGACAAGGGGATACCATTCATGAAGATGAATATCTCGGTCATA ATGCTGCTGGTCAGTGGCTGGAATTCAATAACAGTGATGCCTCTGCTCTGT GGCATAGGGTGCGCCATGCTCCACTGGTCTCTCATTTTACCTGGAATCAAA GCGCAGCAGTCAAAGCTTGCACAGAGAAGGGTGTTCCATGGCGTTGCCAAG AACCCTGTGGTTGATGGGAATCCAACAGTTGACATTGAGGAAGCTCCTGAA ATGCCTGCCCTTTATGAGAAGAAACTGGCTCTATATCTCCTTCTTGCTCTC AGCCTAGCTTCTGTTGCCATGTGCAGAACGCCCTTTTCATTGGCTGAAGGC ATTGTCCTAGCATCAGCTGCCTTAGGGCCGCTCATAGAGGGAAACACCAGC CTTCTTTGGAATGGACCCATGGCTGTCTCCATGACAGGAGTCATGAGGGGG AATCACTATGCTTTTGTGGGAGTCATGTACAATCTATGGAAGATGAAAACT GGACGCCGGGGGAGCGCGAATGGAAAAACTTTGGGTGAAGTCTGGAAGAGG GAACTGAATCTGTTGGACAAGCGACAGTTTGAGTTGTATAAAAGGACCGAC ATTGTGGAGGTGGATCGTGATACGGCACGCAGGCATTTGGCCGAAGGGAAG GTGGACACCGGGGTGGCGGTCTCCAGGGGGACCGCAAAGTTAAGGTGGTTC CATGAGCGTGGCTATGTCAAGCTGGAAGGTAGGGTGATTGACCTGGGGTGT GGCCGCGGAGGCTGGTGTTACTACGCTGCTGCGCAAAAGGAAGTGAGTGGG GTCAAAGGATTTACTCTTGGAAGAGACGGCCATGAGAAACCCATGAATGTG CAAAGTCTGGGATGGAACATCATCACCTTCAAGGACAAAACTGATATCCAC CGCCTAGAACCAGTGAAATGTGACACCCTTTTGTGTGACATTGGAGAGTCA TCATCGTCATCGGTCACAGAGGGGGAAAGGACCGTGAGAGTTCTTGATACT GTAGAAAAATGGCTGGCTTGTGGGGTTGACAACTTCTGTGTGAAGGTGTTA GCTCCATACATGCCAGATGTTCTTGAGAAACTGGAATTGCTCCAAAGGAGG TTTGGCGGAACAGTGATCAGGAACCCTCTCTCCAGGAATTCCACTCATGAA ATGTACTACGTGTCTGGAGCCCGCAGCAATGTCACATTTACTGTGAACCAA ACATCCCGCCTCCTGATGAGGAGAATGAGGCGTCCAACTGGAAAAGTGACC CTGGAGGCTGACGTCATCCTCCCAATTGGGACACGCAGTGTTGAGACAGAC AAGGGACCCCTGGACAAAGAGGCCATAGAAGAAAGGGTTGAGAGGATAAAA TCTGAGTACATGACCTCTTGGTTTTATGACAATGACAACCCCTACAGGACC TGGCACTACTGTGGCTCCTATGTCACAAAAACCTCAGGAAGTGCGGCGAGC ATGGTAAATGGTGTTATTAAAATTCTGACATATCCATGGGACAGGATAGAG GAGGTCACCAGAATGGCAATGACTGACACAACCCCTTTTGGACAGCAAAGA GTGTTTAAAGAAAAAGTTGACACCAGAGCAAAGGATCCACCAGCGGGAACT AGGAAGATCATGAAAGTTGTCAACAGGTGGCTGTTCCGCCACCTGGCCAGA GAAAAGAGCCCCAGACTGTGCACAAAGGAAGAATTTATTGCAAAAGTCCGA AGTCATGCAGCCATTGGAGCTTACCTGGAAGAACAAGAACAGTGGAAGACT GCCAATGAGGCTGTCCAAGACCCAAAGTTCTGGGAACTGGTGGATGAAGAA AGGAAGCTGCACCAACAAGGCAGGTGTCGGACTTGTGTGTACAACATGATG GGGAAAAGAGAGAAGAAGCTGTCAGAGTTTGGGAAAGCAAAGGGAAGCCGT GCCATATGGTATATGTGGCTGGGAGCGCGGTATCTTGAGTTTGAGGCCCTG GGATTCCTGAATGAGGACCATTGGGCTTCCAGGGAAAACTCAGGAGGAGGA GTGGAAGGCATTGGCTTACAATACCTAGGATATGTGATCAGAGACCTGGCT GCAATGGATGGTGGTGGATTCTACGCGGATGACACCGCTGGATGGGACACG CGCATCACAGAGGCAGACCTTGATGATGAACAGGAGATCTTGAACTACATG AGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATGACATACAAG AACAAAGTGGTGAAAGTGTTGAGACCAGCCCCAGGAGGGAAAGCCTACATG GATGTCATAAGTCGACGAGACCAGAGAGGATCCGGGCAGGTAGTGACTTAT GCTCTGAACACCATCACCAACTTGAAAGTCCAATTGATCAGAATGGCAGAA GCAGAGATGGTGATACATCACCAACATGTTCAAGATTGTGATGAATCAGTT CTGACCAGGCTGGAGGCATGGCTCACTGAGCACGGATGTAACAGACTGAAG AGGATGGCGGTGAGTGGAGACGACTGTGTGGTCCGGCCCATCGATGACAGG TTCGGCCTGGCCCTGTCCCATCTCAACGCCATGTCCAAGGTTAGAAAGGAC ATATCTGAATGGCAGCCATCAAAAGGGTGGAATGATTGGGAGAATGTGCCC TTCTGTTCCCACCACTTCCATGAACTACAGCTGAAGGATGGCAGGAGGATT GTGGTGCCTTGCCGAGAACAGGACGAGCTCATTGGGAGAGGAAGGGTGTCT CCAGGAAACGGCTGGATGATCAAGGAAACAGCTTGCCTCAGCAAAGCCTAT GCCAACATGTGGTCACTGATGTATTTTCACAAAAGGGACATGAGGCTACTG TCATTGGCTGTTTCCTCAGCTGTTCCCACCTCATGGGTTCCACAAGGACGC ACAACATGGTCGATTCATGGGAAAGGGGAGTGGATGACCACGGAAGACATG CTTGAGGTGTGGAACAGAGTATGGATAACCAACAACCCACACATGCAGGAC AAGACAATGGTGAAAAAATGGAGAGATGTCCCTTATCTAACCAAGAGACAA GACAAGCTGTGCGGATCACTGATTGGAATGACCAATAGGGCCACCTGGGCC TCCCACATCCATTTAGTCATCCATCGTATCCGAACGCTGATTGGACAGGAG AAATACACTGACTACCTAACAGTCATGGACAGGTATTCTGTGGATGCTGAC CTGCAACTGGGTGAGCTTATCTGAAACACCATCTAACAGGAATAACCGGGA TACAAACCACGGGTGGAGAACCGGACTCCCCACAACCTGAAACCGGGATAT AAACCACGGCTGGAGAACCGGGCTCCGCACTTAAAATGAAACAGAAACCGG GATAAAAACTACGGATGGAGAACCGGACTCCACACATTGAGACAGAAGAAG TTGTCAGCCCAGAACCCCACACGAGTTTTGCCACTGCTAAGCTGTGAGGCA GTGCAGGCTGGGACAGCCGACCTCCAGGTTGCGAAAAACCTGGTTTCTGGG ACCTCCCACCCCAGAGTAAAAAGAACGGAGCCTCCGCTACCACCCTCCCAC GTGGTGGTAGAAAGACGGGGTCTAGAGGTTAGAGGAGACCCTCCAGGGAAC AAATAGTGGGACCATATTGACGCCAGGGAAAGACCGGAGTGGTTCTCTGCT TTTCCTCCAGAGGTCTGTGAGCACAGTTTGCTCAAGAATAAGCAGACCTTT GGATGACAAACACAAAACCAC 3'

[0064] SEQ. ID. NO.: 8 is a precursory sequence of the polyprotein of virus rYF17D/SIVGag45-269. The recombinant protein is shaded in gray.

TABLE-US-00015 SEQ. ID. NO.: 8 SGRKAQGKTLGVNMVRRGVRSLSNKIKQKTKQIGNRPGPSRGVQGFIFFF LFNILTGKKITAHLKRLWKMLDPRQGLAVLRKVKRVVASLMRGLSSRKRR SHDVLTVQFLILGMLLMTGGVTLVRKNRWLLLNVTSEDLGKTFSVGTGNC TTNILEAKYWCPDSMEYNCPNLSPREEPDDIDCWCYGVENVRVAYGKCDS AGRSRRSRRAIDLPTHENHGLKTRQEKWMTGRMGERQLQKIERWFVRNPF FAVTALTIAYLVGSNMTQRVVIALLVLAVGPAYSAHCIGITDRDFIEGVH GGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEARKVCYNAVLT HVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGSIVAC AKFTCAKSMSLFEVDQTKIQYVIRAQLHVGAKQENWNTSIKTLKFDALSG SQEVEFIGYGKATLECQVQTAVDFGNSYIAEMETESWIVDRQWAQDLTLP WQSGSGGVWREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTK DTNDNNLYKLHGGHVSCRVKLSALTLKGTSYKICTDKMFFVKNPTDTGHG TVVMQVKVSKGTPCRIPVIVADDLTAAINKGILVTVNPIASTNDDEVLIE VNPPFGDSYTIVGRGDSRLTYQWHKEGSSIGKLFTQTMKGVERLAVMGDT AWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIMGAVLIWVGI ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## GDGIFIFRDSDDWLNKYSYYPEDPVKLASIVKASFEEGKCGLNSVDSLEH EMWRSRADEINTIFEENEVDISVVVQDPKNVYQRGTHPFSRIRDGLQYGW KTWGKNLVFSPGRKNGSFIIDGKSRKECPFSNRVWNSFQIEEFGTGVETT RVYMDAVFEYTIDCDGSILGAAVNGKKSAHGSPTFWMGSHEVNGTWMIHT LEALDYKECEWPLTHTIGTSVEESEMFMPRSIGGPVSSHNHIPGYKVQTN GPWMQVPLEVKREACPGTSVIIDGNCDGRGKSTRSTTDSGKVIPEWCCRS CTMPPVSFHGSDGCWYPMEIRPRKTHESHLVRSWVTAGEIHAVPFGLVSM MIAMEVVLRKRQGPKQMLVGGVVLLGAMLVGQVTLLDLLKLTVAVGLHFH EMNNGGDAMYMALIAAFSIRPGLLIGFGLRTLWSPRERLVLTLGAAMVEI ALGGVMGGLWKYLNAVSLCILTINAVASRKASNTILPLMALLTPVTMAEV RLAAMFFCAMVIIGVLHQNFKDTSMQKTIPLVALTLTSYLGLTQPFLGLC AFLATRIFGRRSIPVNEALAAAGLVGVLAGLAFQEMENFLGPIAVGGLLM MLVSVAGRVDGLELKKLGEVSWEEEAEISGSSARYDVALSEQGEFKLLSE EKVPWDQVVMTSLALVGAALHPFALLLVLAGWLFHVRGARRSGDVLWDIP TPKIIEECEHLEDGIYGIFQSTFLGASQRGVGVAQGGVFHTMWHVTRGAF LVRNGKKLIPSWASVKEDLVAYGGSWKLEGRWDGEEEVQLIAAVPGKNVV NVQTKPSLFKVRNGGEIGAVALDYPSGTSGSPIVNRNGEVIGLYGNGILV GDNSFVSAISQTEVKEEGKEELQEIPTMLKKGMTTVLDFHPGAGKTRRFL PQILAECARRRLRTLVLAPTRVVLSEMKEAFHGLDVKFHTQAFSAHGSGR EVIDAMCHATLTYRMLEPTRVVNWEVIIMDEAHFLDPASIAARGWAAHRA RANESATILMTATPPGTSDEFPHSNGEIEDVQTDIPSEPWNTGHDWILAD KRPTAWFLPSIRAANVMAASLRKAGKSVVVLNRKTFEREYPTIKQKKPDF ILATDIAEMGANLCVERVLDCRTAFKPVLVDEGRKVAIKGPLRISASSAA QRRGRIGRNPNRDGDSYYYSEPTSENNAHHVCWLEASMLLDNMEVRGGMV APLYGVEGTKTPVSPGEMRLRDDQRKVFRELVRNCDLPVWLSWQVAKAGL KTNDRKWCFEGPEEHEILNDSGETVKCRAPGGAKKPLRPRWCDERVSSDQ SALSEFIKFAEGRRGAAEVLVVLSELPDFLAKKGGEAMDTISVFLHSEEG SRAYRNALSMMPEAMTIVMLFILAGLLTSGMVIFFMSPKGISRMSMAMGT MAGCGYLMFLGGVKPTHISYVMLIFFVLMVVVIPEPGQQRSIQDNQVAYL IIGILTLVSAVAANELGMLEKTKEDLFGKKNLIPSSASPWSWPDLDLKPG AAWTVYVGIVTMLSPMLHHWIKVEYGNLSLSGIAQSASVLSFMDKGIPFM KMNISVIMLLVSGWNSITVMPLLCGIGCAMLHWSLILPGIKAQQSKLAQR RVFHGVAKNPVVDGNPTVDIEEAPEMPALYEKKLALYLLLALSLASVAMC RTPFSLAEGIVLASAALGPLIEGNTSLLWNGPMAVSMTGVMRGNHYAFVG VMYNLWKMKTGRRGSANGKTLGEVWKRELNLLDKRQFELYKRTDIVEVDR DTARRHLAEGKVDTGVAVSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGW CYYAAAQKEVSGVKGFTLGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEP VKCDTLLCDIGESSSSSVTEGERTVRVLDTVEKWLACGVDNFCVKVLAPY MPDVLEKLELLQRRFGGTVIRNPLSRNSTHEMYYVSGARSNVTFTVNQTS RLLMRRMRRPTGKVTLEADVILPIGTRSVETDKGPLDKEAIEERVERIKS EYMTSWFYDNDNPYRTWHYCGSYVTKTSGSAASMVNGVIKILTYPWDRIE EVTRMAMTDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVNRWLFRHLA REKSPRLCTKEEFIAKVRSHAATGAYLEEQEQWKTANEAVQDPKFWELVD EERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMWLGARYLEF EALGFLNEDHWASRENSGGGVEGIGLQYLGYVIRDLAAMDGGGFYADDTA GWDTRITEADLDDEQEILNYMSPHHKKLAQAVMEMTYKNKVVKVLRPAPG GKAYMDVISRRDQRGSGQVVTYALNTITNLKVQLIRMAEAEMVIHHQHVQ DCDESVLTRLEAWLTEHGCNRLKRMAVSGDDCVVRPIDDRFGLALSHLNA MSKVRKDISEWQPSKGWNDWENVPFCSHHFHELQLKDGRRIVVPCREQDE LIGRGRVSPGNGWMIKETACLSKAYANMWSLMYFHKRDMRLLSLAVSSAV PTSWVPQGRTTWSIHGKGEWMTTEDMLEVWNRVWITNNPHMQDKTMVKKW RDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIGQEKYTDYL TVMDRYSVDADLQLGEL

[0065] Other appropriate sequences to prepare the recombinant yellow fever viruses contemplated herein include corresponding sequences in HIV (for example, complete Gag polypeptide or a fragment thereof). For example, the heterologous polypeptide expressed by the recombinant yellow fever viruses considered here may comprise a fragment of SEQ. ID. NO.: 9, or a related protein (for example, a protein having at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of amino acid sequence identity with SEQ. ID. NO.: 9).

TABLE-US-00016 SED. [sic] ID. NO.: 9 MGARASVLSG GKLDKWEKIR LRPGGKKTYQ LKHIVWASRE LERFAVNPGL LETGGGCKQI LVQLQPSLQT GSEELKSLYN AVATLYCVHQ GIEVRDTKEA LDKIEEEQNK SKKKAQQAAA DTGNSSQVSQ NYPIVQNLQG QMVHQAISPR TLNAWVKVIE EKAFSPEVIP MFSALSEGAT PQDLNTMLNT VGGHQAAMQM LKETINEEAA EWDRLHPAHA GPNAPGQMRE PRGSDIAGTT STLQEQIGWM TSNPPVPVGE IYKRWIILGL NKIVRMYSPV SILDIRQGPK EPFRDYVDRF YKTLRAEQAS QDVKNWMTET LLVQNANPDC KTILKALGPA ATLEEMMTAC QGVGGPSHKA RILAEAMSQV TSPANIMMQR GNFRNQRKTI KCFNCGKEGH LARHCRAPRK KGCWKCGREG HQMKDCTERQ ANFLGKIWPS HKGRPGNFLQ SRPEPTAPPE ESFRFGEETT TPPQKQEPLP SQKQETIDKD LYPLASLKSL FGNDPSLQ formatting

[0066] A fragment, as considered here, may include at least 8, 9, 10, 20, 30, 40, 50 [sic] 100, or 200 contiguous amino acids of SEQ. ID. NO.: 9. In one embodiment, the fragment comprises amino acids 45-269 of SEQ. ID. NO.: 9. The heterologous polypeptide may still comprise a terminal amino acid sequence comprising KESSIG.

EXAMPLES

[0067] The present invention will now be described in detail by the examples presented below. It is worth emphasizing that the invention is not limited to these examples, and also includes variations and modifications within the limits allowing its action.

Example 1

Proliferation Properties of Recombinant rYF17D/SIVGag45-269 In Vero Cells

[0068] The growth capacity of recombinant virus rYF17D/SIVGag45-269 was assessed and compared with other two viruses, YF 17DD and YF17D/G1/2 T3 vaccine (the latter corresponding to the parental vector virus, that is, heterologous insertion). Three independent experiments of virus growth in monolayers of Vero cells were performed. All experiments were performed in low MOI of 0.02 with densities of Vero cells of 62,500 cells per cm.sup.2.

[0069] The recombinant virus rYF17D/SIVGag45-269 presented a lower growth rate compared with the viruses of 17D vaccine, and had its peak at 72 hours after the infection, exhibiting a titer of 6.61.+-.0.23 log 10 PFU/mL. FIG. 1 shows the viral growth curves in Vero cells. Cells were infected with YF 17D control (gray lozenges), YF17D/G1/2T3 virus (black squares), or with the recombinant virus rYF17D/SIVGag45-269 (gray triangles in MOI of 0.02). Each time point represents the mean titer obtained for these three experiments separately, with the respective standard deviations.

Table 8 below shows the viral growth curves in Vero cells. Viral titers (log 10 PFU/mL) are shown in each time point. Viral growth peaks are shown in bold.

TABLE-US-00017 TABLE 8 Virus 24 h 48 h 72 h 96 h 120 h 144 h YF17D/17DD 3.60 .+-. 0.54 6.14 .+-. 0.49 7.62 .+-. 1.02 7.63 .+-. 1.03 6.97 .+-. 0.62 7.30 .+-. 0.59 YF/7D/G1/2T3 5.32 .+-. 0.53 7.60 .+-. 1.03 7.45 .+-. 0.33 7.19 .+-. 0.27 6.95 .+-. 0.27 7.23 .+-. 0.40 rYF 17D SIV Gag 3.74 .+-. 0.35 5.57 .+-. 0.51 6.61 .+-. 0.23 6.30 .+-. 0.45 6.04 .+-. 0.27 5.61 .+-. 0.64 45-269

Example 2

Preliminary Immunological Studies of the YF17D Vaccine Virus Infection in Mamu-A*01-Positive Rhesus Monkeys

[0070] First, the YF17D vaccine virus was used to infect four Mamu-A*01-positive monkeys. The vaccine virus replicated in these four animals and induced neutralizing antibodies in all four monkeys by two weeks after vaccination (FIGS. 2A and 2B). To monitor the CD8+ T-cell immune response against YF17D, its proteome was scanned for peptides that might bind to Mamu-A*01 using algorithms (MHC Pathway) [14]. The 52 YF17D-derived peptides which were most likely to bind to Mamu-A*01, based on their predicted affinity for this MHC class I molecule, were synthesized. IFNy ELISPOT assay was used to screen these peptides in YF17D-infected animals, and four Mamu-A*01-binding peptides: 17D 7844 (LTPVTMAEV; LV91285-1293), 17D 7846 (VSPGNGWMI; V193250-3258), 17D 7850 (MSPKGISRM; MM92179-2187), and 17D 7858 (TTPFGQQRVF; TF102853-2863) were recognized in vivo (FIG. 2C). Using a previously reported protocol [15], the activation of CD8+ T cells was also observed in all four animals (FIGS. 2D and 2E). Thus, as previously observed, the YF17D vaccine virus replicates in Indian rhesus monkeys [16] and induces neutralizing antibodies, yellow fever-specific Mamu-A*01-restricted CD8+ T-cell responses, and CD8+ T-cell activation. FIG. 2 shows that YF17D replicates and induces neutralizing antibodies, virus-specific CD8+ T cells, and CD8+ T-cell activation in rhesus monkeys. FIG. 2A shows the replication of YF17D during the first 10 days after vaccination with two different doses, as measured by Q-PCR using YF17D-specific primers. FIG. 2B shows the titer of neutralizing antibodies determined at 2 and 5 weeks after YF17D vaccination. In FIG. 2C, the fresh PBMC from vaccinees (100,000 cells/well) were used in IFN-y ELISPOT assays [17] to assess T-cell responses against YF17D. Four epitopes (17D 7844, 17D 7846, 17D 7850, and 17D 7858)--predicted to bind to Mamu-A*01 as defined by the algorithm (MHC Pathway)--were selected for further studies. FIG. 2D presents the identification of activated CD8+ T cells after vaccination with YF17D based on the expression of proliferation and proapoptotic markers Ki-67 and Bcl-2 [15]. Whole blood cells were stained with antibodies against CD3 and CD8, and then permeabilized, and subsequently labeled with Bcl-2- and Ki-67-specific antibodies. The flow cytometry graphs were gated on CD3+ and CD8+ lymphocytes. FIG. E reveals the expression kinetics of Ki-67 and Bcl-2 in T cells after vaccination with YF17D.

Example 3

Immunogenicity of rYF17D/SIVGag45-269 in Rhesus Monkeys

[0071] In the following step, the YF17D vaccine virus was engineered to express amino acids 45-269 of SIVmac239Gag (rYF17D/SIVGag45-269) by inserting a yellow fever codon-optimized sequence between the genes encoding the viral proteins E and NS1. This recombinant virus replicated and induced neutralizing antibodies in mice (data not shown). The rYF17D/SIVGag45-269 construct was then tested in six Mamu-A*01-positive Indian rhesus monkeys. Evidence for the viral replication of rYF17D/SIVGag45-269 was found for five of these six monkeys (FIG. 3A). Neutralizing antibodies were evident at two weeks after vaccination (FIG. 3B. [sic] A single immunization with rYF17D/SIVGag45-269 induced antigen-specific CD8+ T cells in five out of the six Mamu-A*01-positive monkeys (FIG. 3C). This recombinant virus also induced CD8+ T-cell activation in the majority of the vaccinated animals (FIG. 3D). The sixth monkey (r04091) required a booster dose 28 days after the first immunization to induce specific CD8+ T cells (data not shown and FIG. 3C). No differences were found in vaccine-induced immune responses between the animals vaccinated with YF17D and those vaccinated with rYF17D/SIVGag45-269. There was, however, considerable animal-to-animal variability. Animal r02034, vaccinated with YF17D, exhibited massive CD8+ T-cell activation (a peak of 35% at day 12; FIG. 2E), which was probably induced by the high levels of viral replication (16,800 copies/mL at day 5; FIG. 2A). The comparison of neutralizing antibody responses at 2 and 5 weeks after vaccination in animals vaccinated with YF17D or rYF17D/SIVGag45-269 did not reveal detectable differences regarding the capacity of these two vaccines to induce neutralizing antibodies (FIGS. 2B and 3B). It was also difficult to detect differences in YF17D-specific CD8+ T-cell responses induced by these two vaccines. Peak Mamu-A*01-restricted CD8+ T-cell responses against YF17D ranged from barely detectable (r02110 at day 24; FIG. 2C) to 400 SFCs/10.sup.6 PBMC (r04113 at day 12; FIG. 2C). Two of the animals that were vaccinated with rYF17D/SIVGag45-269 did not originate CD8+ T-cell responses against the Mamu-A*01-bound YF17D peptides, whereas this kind of response was observed in four animals (FIG. 3C), with SFCs/10.sup.6 PBMC ranging from 50 to 200. For almost every animal vaccinated with rYF17D/SIVGag45-269, the Gag CM9-specific responses (peak in 50-750) were higher than those generated against the Mamu-A*01-restricted YF17D epitopes (peak in 0-200), suggesting that the recombinant virus replicated in a stable manner in vivo. Thus, the recombinant YF17D virus replicated and induced both virus-specific neutralizing antibodies and CD8+ T cells that were not demonstrably different from those induced by YF17D alone.

[0072] FIG. 3 shows that rYF17D/SIVGag45-269 replicates and induces neutralizing antibodies, virus-specific CD8+ T cells, and CD8+ T-cell activation in rhesus monkeys. FIG. 3A shows the replication of rYF17D/SIVGag45-269 during the first 10 days after vaccination with two different doses, as measured by Q-PCR using YF17D-specific primers. FIG. 3B shows the titer of neutralizing antibodies determined at 2 and 5 weeks after vaccination with rYF17D/SIVGag45-269. In FIG. 3C, fresh PBMC from vaccinees (100,00[sic] cells/well) were used in ELISPOT-IFN-.gamma. assays to assess T-cell responses against the YF17D vector and SIV Gag (45-269) insert. YF17D-specific responses were measured using the same epitopes described in FIG. 2. For SIV Gag-specific responses, 6 sets of peptides of 15 mer and with 11 mer overlapping were used, covering the whole extension of Gag 45-269 insert of SIVmac239. In addition, Mamu-A*01-restricted responses against the dominant GagCM9181-189 and subdominant GagQI9254-262 epitopes were measured. The peak of CD8+ T-cell responses in r04091 is depicted at 10 days after the second administration of rYF17D/SIVGag (2.0.times.10.sup.5 PFU), which took place on day 28 after the initial vaccination. FIG. 3D shows the expression kinetics of Ki-67 and Bcl-2 in CD8+ T cells after vaccination with rYF17D/SIVGag45-269, as described in FIG. 2.

Example 4

Immunization of Rhesus Monkeys with an Initial Dose (Prime) of Recombinant Mycobacterium bovis BCG Followed by a Booster Dose ("boost") of rYF17D/SIVGag45-269 Virus

[0073] Most viral vectors are usually more efficient after an initial dose [prime] with DNA or rBCG [18, 19, 20, 21]. Thus, two monkeys that had been primed with rBCG expressing SIV proteins (FIG. 4A) were given a booster dose. No SIV-specific response was detected after either of the two priming rBCG vaccinations. Unfortunately, only a low level of rYF17D/SIVGag45-269 replication was observed at day 5 after vaccination in one of the rBCG-primed animals (r01108, 7 copies/mL; FIG. 4B). However, both animals generated neutralizing antibodies at 2 weeks after vaccination (FIG. 4C). Fortunately, high-frequency CD8+ T-cell responses were detected in the Mamu-A*01-positive monkey (r01056) after a booster dose with rYF17D/SIVGag45-269 (FIGS. 4D and 4E). The booster dose induced massive activation of CD8+ T cells in the Mamu-A*01-positive animal r01056, peaking at 35% at 17 days after vaccination (FIGS. 4D and 4E). A frequency of 3.5% of the CD8+ T cells of this animal responded to the Mamu-A*01 GagCM9181-189 epitope at day 17 after vaccination (FIGS. 4D and 4E). These CD8+ T cells synthesized IFN .gamma., TNF.alpha., MIP-1.beta. and degranulated (FIG. 4E and data not shown).

[0074] Thus, an rBCG prime followed by a recombinant yellow fever 17D boost induced polyfunctional antigen-specific CD8+ T cells.

[0075] Vaccine-induced CD8+ T cells may be central memory T cells (TCM) or effector memory T cells (TEM). These two subsets of CD8+ T cells differ in function and surface markers [22]. Repeated boosting drives CD8+ T cells toward the TEM subset [22]. Consequently, we evaluated whether an initial dose of rBCG followed by a booster dose of rYF17D/SIVGag45-269 induced TCM or TEM CD8+ T cells. Staining revealed that the SIV-specific CD8+ T cells were largely TEM cells since most of them were CD28+(FIG. 5A). It was recently suggested that TEM cells residing in mucosas can effectively control infection after a low-dose challenge with SIVmac239 [23].

[0076] After that, rYF17D/SIVGag45-269-induced CD8+ T cells were tested so as to determine if they could recognize virally infected CD8+ T cells. It was shown that CD8+ T cells stain for tetramers and produce cytokines after stimulation with synthetic peptides. Nevertheless, none of these assays determines whether these CD8+ T cells may recognize SIV-infected cells and reduce viral replication. Thus, a recently developed assay [24] was used to determine whether vaccine-induced CD8+ T cells can reduce viral replication in CD4+ T cells. Sorted tetramer(-) (GagCM9181-189) lymphocytes were incubated for 48 hours with SIVmac239-infected CD4+ T cells that expressed Mamu-A*01. The number of CD4+ T cells that expressed SIV Gag and the quantity of virus in the culture supernatant were assessed. Vaccine-induced CD8+ T cells reduced viral replication to the same extent as that seen with purified SIV-specific CD8+ T cells from three SIVmac239-infected rhesus monkeys, including an elite controller rhesus monkey 95061 (FIG. 5B).

[0077] Thus, in one aspect of the methods disclosed here, it is important that the rBCG has induced a high-frequency CD8+ T-cell response after a booster dose of rYF17D/SIVGag45-269. These CD8+ T cells reached frequencies that were similar to those induced by an initial dose of rBCG followed by a booster dose of Ad5 [19]. Even without the benefit of the initial dose of rBCG, the levels of CD8+ T cells induced by a single rYF17D/SIVGag45-269 vaccination were equivalent to those induced by our best SIV vaccine, SIVmac239ANef. Recombinant YF17D generated an average of 195 (range of 100-750) SFC/10 6 [sic] PBMC (N=6), whereas SIVmac239ANef induced an average of 238 (range of 150-320) SFC/10.sup.6 PBMC (N=3) [25]. It is also possible that any YF17D/HIV recombinants would likely replicate better in humans than in rhesus monkeys, thus inducing more robust immune responses. The rBCG was shown to be more effective in humans [26, 27, 28, 29] and may be more useful for inducing T-cell responses in humans than it has been in our limited study with rhesus monkeys.

[0078] FIG. 4 shows that vaccination with rYF17D/SIVGag45-269 induced a robust expansion of Gag-specific responses in an rBCG-primed monkey. FIG. 4A shows the vaccination scheme. Two rhesus monkeys were immunized with rBCG intradermally (i.d.) (2.times.10.sup.5 CFU), rBCG orally (10.sup.9 CFU), and rYF17D/SIVGag45-269 subcutaneously (2.times.10.sup.5 PFU) at 6-month intervals. The rBCG was engineered to express 18 minigenes containing sequences of Gag, Vif, Nef, Ver, and Tat from SIVmac239. (See Tables 2-6, Proteins 1(a), 2(a), 3(a), 4(a), 6(a), 8(a), 12(a), 13(a), 14(a), 15(a), 17(a), 18 (a), 19(a), 20 (a), 21 (a), 22 (a), 23(a), e 24(a)). FIG. 4B shows the replication of rYF17D/SIVGag45-269 during the first 10 days after vaccination, as measured by Q-PCR using YF17D-specific primers. FIG. 4C shows the titer of neutralizing antibodies determined at 2 and 5 weeks after vaccination with rYF17D/SIVGag45-269. FIG. 4D shows the kinetics of CD8+ T-cell activation (as described in FIG. 1) and expansion of CM9181-189-specific CD8+ T cells in r01056 after vaccination with rYF17D/SIVGag45-269. In FIG. 4E, it can be seen that vaccination with rYF17D/SIVGag45-269 induces robust CD8+ T-cell responses against GagCM9181-189 in r01056. CD8+ T-cell activation (Ki-67+/Bcl-2-[sic]) for baseline and day 13 are shown. GagCM9181-189-specific responses were measured by tetramer staining, intracellular cytokine staining with antibodies against MIP-1.beta. and IFN-.gamma., and ELISPOT-IFN-.gamma..

[0079] FIG. 5 illustrates that rYF17D/SIVGag45-269 vaccination of r01056 provides effector memory GagCM9181-189-specific CD8+ T cells that suppresses viral replication in CD4+targets. FIG. 5A shows the frequency of tetramer-positive GagCM9181-189-specific CD8+ T cells in r01056 gated on CD3+CD8+lymphocytes. In FIG. 5B, CD28 and CD95 expression profiles of tetramer-positive cells show a polarized effector memory phenotype. FIG. 5C shows that ex vivo GagCM9181-189-specific CD8+ T cells from r01056 inhibit viral replication from SIVmac239-infected CD4+ T cells. GagCM9181-189-specific CD8+ T cells of three SIV-infected Manu-A*01-positive animals and r01056 vaccinated with rYF17D/SIVGag45-269 were tested for their ability to suppress viral replication from SIV-infected CD4+ T cells [24].

[0080] Forty-eight hours after the incubation of various samples containing different ratios of SIV-infected CD4+ T cells and GagCM9191-189-specific CD8+ T cells, the supernatant of these samples was removed and measured for viral RNA copies per milliliter by Q-PCR. No suppression was observed when effectors were incubated with CD4+ targets from Manu-A*01-negative animals. Rh2021 was infected with SIVmac239 (viral load .about.10.sup.5 vRNA copies/mL) containing mutations in 8 Manu-B*08-restricted epitopes as part of another study [30]. R01080 was vaccinated with a DNA/Ad5 regimen expressing gag, Rev, Tat and Nef, and later infected with SIVmac239 (viral load .about.10.sup.3 vRNA copies/mL) [31]. R95061 was vaccinated with a DNA/MVA regimen containing Gag CM9181-189 and was later challenged with SIVmac239 (undetectable viral load) [32].

[0081] From the facts exposed above, one may conclude that the attenuated yellow fever vaccine virus 17D expressing SIVmac239 Gag, as described herein, provided excellent immunization responses.

[0082] The material above is illustrative of the present invention, and is not intended to limit the scope of the claimed subject matter. The invention is defined by the claims below.

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A. Jarvis, and L. J. Picker. 2009. Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat Med 15:293-299. [0106] [24]--Vojnov, L. J. Reed, K. L. Weisgrau, E. Rakasz, J. T. Loffredo, S. Piaskowski, J. B. Sacha, H. L. Kolar, N. A. Wilson, R. P. Johnson, and D. I. Watkins. Effective SIV-specific CD8+ T-cells lack an easily detectable, shared characteristic. J Virol Manuscript in press. [0107] [25]--Reynolds, M R, A. M. Weiler, K. L. Weisgrau, S. M. Piaskowski, J. R. Furlott, J. T. Weinfurther, M Kaizu, T. Soma, E. J. Leon, C. MacNair, D. P. Leaman, M. B. Zwick, E. Gostick, S. K. Musani, D. A. Price, T. C. Friedrich, E. G. Rakasz, N. A. Wilson, A. B. McDermott, R. Boyle, D. B. Allison, D. R. Burton, W. C. Koff, and D. I. Watkins. 2008. Macaques vaccinated with live-attenuated SIV control replication of heterologous virus. J Exp Med 205:2537-2550. [0108] [26]--Barker, L. F., M. J. Brennan, P. K. Rosenstein, and J. C. Sadoff. 2009. Tuberculosis vaccine research: the impact of immunology. Curr Opin Immunol 21:331-338. [0109] [27]--Hoft, D. F., A. Blazevic, G. Abate, W. A. Hanekom, G. Kaplan, J. H. Soler, F. Weichold, L. Geiter, J. C. Sadoff, and M. A. Horwitz. 2008. A new recombinant bacille Calmette-Guerin vaccine safely induces significantly enhanced tuberculosis-specific immunity in human volunteers. J Infect Dis 198:1491-1501. [0110] [28]--Skeiky, Y. A., and J. C. Sadoff. 2006. Advances in tuberculosis vaccine strategies. Nat Rev Microbiol 4:469-476 [0111] [29]--Stover, C. K., G. P. Bansal, S. Langerman, and M. S. Hanson. 1994. Protective immunity elicited by rBCG vaccines. Dev Biol Stand 82:163-170. [0112] [30]--Valentine, L. E., J. T. Loffredo, A. T. Bean, E. J. Leon, C. E. Macnair, D. R. Beal, S. M. Piaskowski, Y. C. Klimentidis, S. M. Lank, R. W. Wiseman, J. T. Weinfurter, G. E. May, E. G. Rakasz, N. A. Wilson, T. C. Friedrich, D. H. O'Connor, D. B. Allison, and D. I. Watkins. 2009. Infection with "escaped" virus variants impairs control of SIVmac239 replication in Mamu-B*08+macaques. J. Virol., in press. [0113] [31]--Wilson, N. A., J. Reed, G. S, Napoe, S. Piaskowski, A. Szymanski, J. Furlott, E. J Gonzalez, L. J. Yant, N. J. Maness, G. E. May, T. Soma, M. R. Reynolds, E. Rakasz, R. Rudersdorf, A. B. MacDermott, D. H. O'Connor, T. C. Friedrich, D. B. Alison, A. Patki, L. J. Picker, D. R. Burton, J. Lin, L. Huang, D. Patel, G. Heindecker, J. Fan, M. Citron, M. Horton, F. Wang, X. Liang, J. W. Shiver, D. R. Casimiro, and D. I. Watkins. 2006. Vaccine-induced cellular immune responses reduce plasma viral concentrations after repeated low-dose challenge with pathogenic simian immunodeficiency virus SIVmac239. J Virol 80:5875-5885. [0114] [32]--Allen, T. M., P. Jing, B. Calore, H. Horton, D. H. O'Connor, T. Hanke, M. Piekarczyk, R. Ruddersdorf, B. R. Mothe, C. Emerson, N. Wilson, J. D. Lifson, I. M. Belyakov, J. A. Berzofsky, C. Wang, D. B. Allison, D. C. Montefiori, R. C. Desrosiers, S. Wolinsky, K. J. Kunstman, J. D. Altman, A. Sette, A. J. McMichael, and D. I. Watkins. 2002. Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection. J Virol 76:10507-10511.

Sequence CWU 1

1

91510PRTSimian immunodeficiency virusmisc_feature(489)..(489)Xaa can be any naturally occurring amino acid 1Met Gly Ala Arg Asn Ser Val Leu Ser Gly Lys Lys Ala Asp Glu Leu1 5 10 15Glu Lys Ile Arg Leu Arg Pro Asn Gly Lys Lys Lys Tyr Met Leu Lys 20 25 30His Val Val Trp Ala Ala Asn Glu Leu Asp Arg Phe Gly Leu Ala Glu 35 40 45Ser Leu Leu Glu Asn Lys Glu Gly Cys Gln Lys Ile Leu Ser Val Leu 50 55 60Ala Pro Leu Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Tyr Asn65 70 75 80Thr Val Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys His 85 90 95Thr Glu Glu Ala Lys Gln Ile Val Gln Arg His Leu Val Val Glu Thr 100 105 110Gly Thr Thr Glu Thr Met Pro Lys Thr Ser Arg Pro Thr Ala Pro Ser 115 120 125Ser Gly Arg Gly Gly Asn Tyr Pro Val Gln Gln Ile Gly Gly Asn Tyr 130 135 140Val His Leu Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Leu145 150 155 160Ile Glu Glu Lys Lys Phe Gly Ala Glu Val Val Pro Gly Phe Gln Ala 165 170 175Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys 180 185 190Val Gly Asp His Gln Ala Ala Met Gln Ile Ile Arg Asp Ile Ile Asn 195 200 205Glu Glu Ala Ala Asp Trp Asp Leu Gln His Pro Gln Pro Ala Pro Gln 210 215 220Gln Gly Gln Leu Arg Glu Pro Ser Gly Ser Asp Ile Ala Gly Thr Thr225 230 235 240Ser Ser Val Asp Glu Gln Ile Gln Trp Met Tyr Arg Gln Gln Asn Pro 245 250 255Ile Pro Val Gly Asn Ile Tyr Arg Arg Trp Ile Gln Leu Gly Leu Gln 260 265 270Lys Cys Val Arg Met Tyr Asn Pro Thr Asn Ile Leu Asp Val Lys Gln 275 280 285Gly Pro Lys Glu Pro Phe Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser 290 295 300Leu Arg Ala Glu Gln Thr Asp Ala Ala Val Lys Asn Trp Met Thr Gln305 310 315 320Thr Leu Leu Ile Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys 325 330 335Gly Leu Gly Val Asn Pro Thr Leu Glu Glu Met Leu Thr Ala Cys Gln 340 345 350Gly Val Gly Gly Pro Gly Gln Lys Ala Arg Leu Met Ala Glu Ala Leu 355 360 365Lys Glu Ala Leu Ala Pro Val Pro Ile Pro Phe Ala Ala Ala Gln Gln 370 375 380Arg Gly Pro Arg Lys Thr Ile Lys Cys Trp Asn Cys Gly Lys Glu Gly385 390 395 400His Ser Ala Arg Gln Cys Arg Ala Pro Arg Arg Gln Gly Cys Trp Lys 405 410 415Cys Gly Lys Met Asp His Val Met Ala Lys Cys Pro Asp Arg Gln Ala 420 425 430Gly Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe Pro 435 440 445Met Ala Gln Val His Gln Gly Leu Met Pro Thr Ala Pro Pro Glu Asp 450 455 460Pro Ala Val Asp Leu Leu Lys Asn Tyr Met Gln Leu Gly Lys Gln Gln465 470 475 480Arg Glu Lys Gln Arg Glu Ser Arg Xaa Lys Pro Tyr Lys Glu Val Thr 485 490 495Glu Asp Leu Leu His Leu Asn Ser Leu Phe Gly Gly Asp Gln 500 505 5102307PRTSimian immunodeficiency virus 2Asp Gln Gly Ser Ala Ile Asn Phe Gly Arg Gly Leu Ala Glu Ser Leu1 5 10 15Leu Glu Asn Lys Glu Gly Cys Gln Lys Ile Leu Ser Val Leu Ala Pro 20 25 30Leu Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Tyr Asn Thr Val 35 40 45Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys His Thr Glu 50 55 60Glu Ala Lys Gln Ile Val Gln Arg His Leu Val Val Glu Thr Gly Thr65 70 75 80Thr Glu Thr Met Pro Lys Thr Ser Arg Pro Thr Ala Pro Ser Ser Gly 85 90 95Arg Gly Gly Asn Tyr Pro Val Gln Gln Ile Gly Gly Asn Tyr Val His 100 105 110Leu Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Leu Ile Glu 115 120 125Glu Lys Lys Phe Gly Ala Glu Val Val Pro Gly Phe Gln Ala Leu Ser 130 135 140Glu Gly Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys Val Gly145 150 155 160Asp His Gln Ala Ala Met Gln Ile Ile Arg Asp Ile Ile Asn Glu Glu 165 170 175Ala Ala Asp Trp Asp Leu Gln His Pro Gln Pro Ala Pro Gln Gln Gly 180 185 190Gln Leu Arg Glu Pro Ser Gly Ser Asp Ile Ala Gly Thr Thr Ser Ser 195 200 205Val Asp Glu Gln Ile Gln Trp Met Tyr Arg Gln Gln Asn Pro Ile Pro 210 215 220Val Gly Asn Ile Tyr Arg Arg Trp Ile Gln Leu Lys Glu Ser Ser Ile225 230 235 240Gly Thr Ser Leu Gly Lys Ala Val His Gln Val Phe Gly Ser Val Tyr 245 250 255Thr Thr Met Phe Gly Gly Val Ser Trp Met Ile Arg Ile Leu Ile Gly 260 265 270Phe Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr Ser Met Ala 275 280 285Met Thr Cys Ile Ala Val Gly Gly Ile Thr Leu Phe Leu Gly Phe Thr 290 295 300Val Gly Ala305310PRTYellow fever virus 3Asp Gln Gly Ser Ala Ile Asn Phe Gly Arg1 5 1046PRTYellow fever virus 4Lys Glu Ser Ser Ile Gly1 5566PRTDengue virus type 4 5Thr Ser Leu Gly Lys Ala Val His Gln Val Phe Gly Ser Val Tyr Thr1 5 10 15Thr Met Phe Gly Gly Val Ser Trp Met Ile Arg Ile Leu Ile Gly Phe 20 25 30Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr Ser Met Ala Met 35 40 45Thr Cys Ile Ala Val Gly Gly Ile Thr Leu Phe Leu Gly Phe Thr Val 50 55 60Gly Ala656924DNASimian immunodeficiency virus 6gatcaaggaa gcgccatcaa cttcggccga ggactggctg aatcactgct ggaaaacaaa 60gaaggatgtc agaaaatcct gtcagtgctg gctccactgg tgccaacagg atcagaaaac 120ctgaagtcac tgtacaacac agtgtgtgtg atctggtgta tccatgctga agaaaaagtg 180aagcatacag aagaggccaa gcagatcgtg cagaggcatc tggtggtgga aacagggaca 240acagaaacaa tgccaaagac atcaaggcca acagctccat catcaggaag ggggggaaac 300tacccagtgc agcagatcgg agggaactac gtgcatctgc cactgtcacc aaggacactg 360aacgcttggg tgaaactgat cgaagaaaag aaatttggag ctgaagtggt gccaggattt 420caggctctgt cagaaggatg tacaccatac gacatcaacc agatgctgaa ctgtgtggga 480gatcatcagg ctgctatgca gatcattagg gacatcatca acgaagaagc tgcagattgg 540gatctgcaac atccacagcc agctccacag cagggacagc tgagggaacc atcaggatca 600gacattgctg gaacaacatc atcagtggac gaacagatcc agtggatgta caggcagcag 660aacccaatcc cagtgggaaa catctacaga agatggatcc agctgaaaga gggaagctca 720ataggaacat cattgggaaa ggctgtgcac caggtttttg gaagtgtgta tacaaccatg 780tttggaggag tctcatggat gattagaatc ctaattgggt tcttagtgtt gtggattggc 840acgaactcca ggaacacttc aatggctatg acgtgcatag ctgttggagg aatcactctg 900tttctgggct tcacagttgg cgcc 924711785DNASimian immunodeficiency virus 7agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgaccggtgg 480agtgaccttg gtgcggaaaa acagatggtt gctcctaaat gtgacatctg aggacctcgg 540gaaaacattc tctgtgggca caggcaactg cacaacaaac attttggaag ccaagtactg 600gtgcccagac tcaatggaat acaactgtcc caatctcagt ccaagagagg agccagatga 660cattgattgc tggtgctatg gggtggaaaa cgttagagtc gcatatggta agtgtgactc 720agcaggcagg tctaggaggt caagaagggc cattgacttg cctacgcatg aaaaccatgg 780tttgaagacc cggcaagaaa aatggatgac tggaagaatg ggtgaaaggc aactccaaaa 840gattgagaga tggttcgtga ggaacccctt ttttgcagtg acggctctga ccattgccta 900ccttgtggga agcaacatga cgcaacgagt cgtgattgcc ctactggtct tggctgttgg 960tccggcctac tcagctcact gcattggaat tactgacagg gatttcattg agggggtgca 1020tggaggaact tgggtttcag ctaccctgga gcaagacaag tgtgtcactg ttatggcccc 1080tgacaagcct tcattggaca tctcactaga gacagtagcc attgatagac ctgctgaggc 1140gaggaaagtg tgttacaatg cagttctcac tcatgtgaag attaatgaca agtgccccag 1200cactggagag gcccacctag ctgaagagaa cgaaggggac aatgcgtgca agcgcactta 1260ttctgataga ggctggggca atggctgtgg cctatttggg aaagggagca ttgtggcatg 1320cgccaaattc acttgtgcca aatccatgag tttgtttgag gttgatcaga ccaaaattca 1380gtatgtcatc agagcacaat tgcatgtagg ggccaagcag gaaaattgga ataccagcat 1440taagactctc aagtttgatg ccctgtcagg ctcccaggaa gtcgagttca ttgggtatgg 1500aaaagctaca ctggaatgcc aggtgcaaac tgcggtggac tttggtaaca gttacatcgc 1560tgagatggaa acagagagct ggatagtgga cagacagtgg gcccaggact tgaccctgcc 1620atggcagagt ggaagtggcg gggtgtggag agagatgcat catcttgtcg aatttgaacc 1680tccgcatgcc gccactatca gagtactggc cctgggaaac caggaaggct ccttgaaaac 1740agctcttact ggcgcaatga gggttacaaa ggacacaaat gacaacaacc tttacaaact 1800acatggtgga catgtttctt gcagagtgaa attgtcagct ttgacactca aggggacatc 1860ctacaaaata tgcactgaca aaatgttttt tgtcaagaac ccaactgaca ctggccatgg 1920cactgttgtg atgcaggtga aagtgtcaaa aggaaccccc tgcaggattc cagtgatagt 1980agctgatgat cttacagcgg caatcaataa aggcattttg gttacagtta accccatcgc 2040ctcaaccaat gatgatgaag tgctgattga ggtgaaccca ccttttggag acagctacat 2100tatcgttggg agaggagatt cacgtctcac ttaccagtgg cacaaagagg gaagctcaat 2160aggaaagttg ttcactcaga ccatgaaagg cgtggaacgc ctggccgtca tgggagacac 2220cgcctgggat ttcagctccg ctggagggtt cttcacttcg gttgggaaag gaattcatac 2280ggtgtttggc tctgcctttc aggggctatt tggcggcttg aactggataa caaaggtcat 2340catgggggcg gtactcatat gggttggcat caacacaaga aacatgacaa tgtccatgag 2400catgatcttg gtaggagtga tcatgatgtt tttgtctcta ggagttgggg cggatcaagg 2460aagcgccatc aacttcggcc gaggactggc tgaatcactg ctggaaaaca aagaaggatg 2520tcagaaaatc ctgtcagtgc tggctccact ggtgccaaca ggatcagaaa acctgaagtc 2580actgtacaac acagtgtgtg tgatctggtg tatccatgct gaagaaaaag tgaagcatac 2640agaagaggcc aagcagatcg tgcagaggca tctggtggtg gaaacaggga caacagaaac 2700aatgccaaag acatcaaggc caacagctcc atcatcagga agggggggaa actacccagt 2760gcagcagatc ggagggaact acgtgcatct gccactgtca ccaaggacac tgaacgcttg 2820ggtgaaactg atcgaagaaa agaaatttgg agctgaagtg gtgccaggat ttcaggctct 2880gtcagaagga tgtacaccat acgacatcaa ccagatgctg aactgtgtgg gagatcatca 2940ggctgctatg cagatcatta gggacatcat caacgaagaa gctgcagatt gggatctgca 3000acatccacag ccagctccac agcagggaca gctgagggaa ccatcaggat cagacattgc 3060tggaacaaca tcatcagtgg acgaacagat ccagtggatg tacaggcagc agaacccaat 3120cccagtggga aacatctaca gaagatggat ccagctgaaa gagggaagct caataggaac 3180atcattggga aaggctgtgc accaggtttt tggaagtgtg tatacaacca tgtttggagg 3240agtctcatgg atgattagaa tcctaattgg gttcttagtg ttgtggattg gcacgaactc 3300caggaacact tcaatggcta tgacgtgcat agctgttgga ggaatcactc tgtttctggg 3360cttcacagtt ggcgccgatc aaggatgcgc catcaacttt ggcaagagag agctcaagtg 3420cggagatggt atcttcatat ttagagactc tgatgactgg ctgaacaagt actcatacta 3480tccagaagat cctgtgaagc ttgcatcaat agtgaaagcc tctttcgaag aagggaagtg 3540tggcctaaat tcagttgact cccttgagca tgagatgtgg agaagcaggg cagatgagat 3600taataccatt tttgaggaaa acgaggtgga catttctgtt gtcgtgcagg atccaaagaa 3660tgtttaccag agaggaactc atccattttc cagaattcgg gatggtctgc agtatggttg 3720gaagacttgg ggtaagaacc ttgtgttctc cccagggagg aagaatggaa gcttcatcat 3780agatggaaag tccaggaaag aatgcccgtt ttcaaaccgg gtctggaatt ctttccagat 3840agaggagttt gggacgggag tgttcaccac acgcgtgtac atggacgcag tctttgaata 3900caccatagac tgcgatggat ctatcttggg tgcagcggtg aacggaaaaa agagtgccca 3960tggctctcca acattttgga tgggaagtca tgaagtaaat gggacatgga tgatccacac 4020cttggaggca ttagattaca aggagtgtga gtggccactg acacatacga ttggaacatc 4080agttgaagag agtgaaatgt tcatgccgag atcaatcgga ggcccagtta gctctcacaa 4140tcatatccct ggatacaagg ttcagacgaa cggaccttgg atgcaggtac cactagaagt 4200gaagagagaa gcttgcccag ggactagcgt gatcattgat ggcaactgtg atggacgggg 4260aaaatcaacc agatccacca cggatagcgg gaaagttatt cctgaatggt gttgccgctc 4320ctgcacaatg ccgcctgtga gcttccatgg tagtgatggg tgttggtatc ccatggaaat 4380taggccaagg aaaacgcatg aaagccatct ggtgcgctcc tgggttacag ctggagaaat 4440acatgctgtc ccttttggtt tggtgagcat gatgatagca atggaagtgg tcctaaggaa 4500aagacaggga ccaaagcaaa tgttggttgg aggagtagtg ctcttgggag caatgctggt 4560cgggcaagta actctccttg atttgctgaa actcacagtg gctgtgggat tgcatttcca 4620tgagatgaac aatggaggag acgccatgta tatggcgttg attgctgcct tttcaatcag 4680accagggctg ctcatcggct ttgggctcag gaccctatgg agccctcggg aacgccttgt 4740gctgacccta ggagcagcca tggtggagat tgccttgggt ggcgtgatgg gcggcctgtg 4800gaagtatcta aatgcagttt ctctctgcat cctgacaata aatgctgttg cttctaggaa 4860agcatcaaat accatcttgc ccctcatggc tctgttgaca cctgtcacta tggctgaggt 4920gagacttgcc gcaatgttct tttgtgccat ggttatcata ggggtccttc accagaattt 4980caaggacacc tccatgcaga agactatacc tctggtggcc ctcacactca catcttacct 5040gggcttgaca caaccttttt tgggcctgtg tgcatttctg gcaacccgca tatttgggcg 5100aaggagtatc ccagtgaatg aggcactcgc agcagctggt ctagtgggag tgctggcagg 5160actggctttt caggagatgg agaacttcct tggtccgatt gcagttggag gactcctgat 5220gatgctggtt agcgtggctg ggagggtgga tgggctagag ctcaagaagc ttggtgaagt 5280ttcatgggaa gaggaggcgg agatcagcgg gagttccgcc cgctatgatg tggcactcag 5340tgaacaaggg gagttcaagc tgctttctga agagaaagtg ccatgggacc aggttgtgat 5400gacctcgctg gccttggttg gggctgccct ccatccattt gctcttctgc tggtccttgc 5460tgggtggctg tttcatgtca ggggagctag gagaagtggg gatgtcttgt gggatattcc 5520cactcctaag atcatcgagg aatgtgaaca tctggaggat gggatttatg gcatattcca 5580gtcaaccttc ttgggggcct cccagcgagg agtgggagtg gcacagggag gggtgttcca 5640cacaatgtgg catgtcacaa gaggagcttt ccttgtcagg aatggcaaga agttgattcc 5700atcttgggct tcagtaaagg aagaccttgt cgcctatggt ggctcatgga agttggaagg 5760cagatgggat ggagaggaag aggtccagtt gatcgcggct gttccaggaa agaacgtggt 5820caacgtccag acaaaaccga gcttgttcaa agtgaggaat gggggagaaa tcggggctgt 5880cgctcttgac tatccgagtg gcacttcagg atctcctatt gttaacagga acggagaggt 5940gattgggctg tacggcaatg gcatccttgt cggtgacaac tccttcgtgt ccgccatatc 6000ccagactgag gtgaaggaag aaggaaagga ggagctccaa gagatcccga caatgctaaa 6060gaaaggaatg acaactgtcc ttgattttca tcctggagct gggaagacaa gacgtttcct 6120cccacagatc ttggccgagt gcgcacggag acgcttgcgc actcttgtgt tggcccccac 6180cagggttgtt ctttctgaaa tgaaggaggc ttttcacggc ctggacgtga aattccacac 6240acaggctttt tccgctcacg gcagcgggag agaagtcatt gatgccatgt gccatgccac 6300cctaacttac aggatgttgg aaccaactag ggttgttaac tgggaagtga tcattatgga 6360tgaagcccat tttttggatc cagctagcat agccgctaga ggttgggcag cgcacagagc 6420tagggcaaat gaaagtgcaa caatcttgat gacagccaca ccgcctggga ctagtgatga 6480atttccacat tcaaatggtg aaatagaaga tgttcaaacg gacataccca gtgagccctg 6540gaacacaggg catgactgga tcctggctga caaaaggccc acggcatggt tccttccatc 6600catcagagct gcaaatgtca tggctgcctc tttgcgtaag gctggaaaga gtgtggtggt 6660cctgaacagg aaaacctttg agagagaata ccccacgata aagcagaaga aacctgactt 6720tatattggcc actgacatag ctgaaatggg agccaacctt tgcgtggagc gagtgctgga 6780ttgcaggacg gcttttaagc ctgtgcttgt ggatgaaggg aggaaggtgg caataaaagg 6840gccacttcgt atctccgcat cctctgctgc tcaaaggagg gggcgcattg ggagaaatcc 6900caacagagat ggagactcat actactattc tgagcctaca agtgaaaata atgcccacca 6960cgtctgctgg ttggaggcct caatgctctt ggacaacatg gaggtgaggg gtggaatggt 7020cgccccactc tatggcgttg aaggaactaa aacaccagtt tcccctggtg aaatgagact 7080gagggatgac cagaggaaag tcttcagaga actagtgagg aattgtgacc tgcccgtttg 7140gctttcgtgg caagtggcca aggctggttt gaagacgaat gatcgtaagt ggtgttttga 7200aggccctgag gaacatgaga tcttgaatga cagcggtgaa acagtgaagt gcagggctcc 7260tggaggagca aagaagcctc tgcgcccaag gtggtgtgat gaaagggtgt catctgacca 7320gagtgcgctg tctgaattta ttaagtttgc tgaaggtagg aggggagctg ctgaagtgct 7380agttgtgctg agtgaactcc ctgatttcct ggctaaaaaa ggtggagagg caatggatac 7440catcagtgtg ttcctccact ctgaggaagg ctctagggct taccgcaatg cactatcaat 7500gatgcctgag gcaatgacaa tagtcatgct gtttatactg gctggactac tgacatcggg 7560aatggtcatc tttttcatgt ctcccaaagg catcagtaga atgtctatgg cgatgggcac 7620aatggccggc tgtggatatc tcatgttcct tggaggcgtc aaacccactc acatctccta 7680tgtcatgctc atattctttg tcctgatggt ggttgtgatc cccgagccag ggcaacaaag 7740gtccatccaa gacaaccaag tggcatacct cattattggc atcctgacgc tggtttcagc 7800ggtggcagcc aacgagctag gcatgctgga gaaaaccaaa gaggacctct ttgggaagaa 7860gaacttaatt ccatctagtg cttcaccctg gagttggccg gatcttgacc tgaagccagg 7920agctgcctgg acagtgtacg ttggcattgt tacaatgctc tctccaatgt tgcaccactg 7980gatcaaagtc gaatatggca acctgtctct gtctggaata gcccagtcag cctcagtcct 8040ttctttcatg gacaagggga taccattcat gaagatgaat atctcggtca taatgctgct 8100ggtcagtggc tggaattcaa taacagtgat gcctctgctc tgtggcatag ggtgcgccat 8160gctccactgg tctctcattt tacctggaat caaagcgcag cagtcaaagc ttgcacagag 8220aagggtgttc catggcgttg ccaagaaccc tgtggttgat gggaatccaa cagttgacat 8280tgaggaagct cctgaaatgc ctgcccttta tgagaagaaa ctggctctat atctccttct 8340tgctctcagc ctagcttctg ttgccatgtg cagaacgccc ttttcattgg ctgaaggcat

8400tgtcctagca tcagctgcct tagggccgct catagaggga aacaccagcc ttctttggaa 8460tggacccatg gctgtctcca tgacaggagt catgaggggg aatcactatg cttttgtggg 8520agtcatgtac aatctatgga agatgaaaac tggacgccgg gggagcgcga atggaaaaac 8580tttgggtgaa gtctggaaga gggaactgaa tctgttggac aagcgacagt ttgagttgta 8640taaaaggacc gacattgtgg aggtggatcg tgatacggca cgcaggcatt tggccgaagg 8700gaaggtggac accggggtgg cggtctccag ggggaccgca aagttaaggt ggttccatga 8760gcgtggctat gtcaagctgg aaggtagggt gattgacctg gggtgtggcc gcggaggctg 8820gtgttactac gctgctgcgc aaaaggaagt gagtggggtc aaaggattta ctcttggaag 8880agacggccat gagaaaccca tgaatgtgca aagtctggga tggaacatca tcaccttcaa 8940ggacaaaact gatatccacc gcctagaacc agtgaaatgt gacacccttt tgtgtgacat 9000tggagagtca tcatcgtcat cggtcacaga gggggaaagg accgtgagag ttcttgatac 9060tgtagaaaaa tggctggctt gtggggttga caacttctgt gtgaaggtgt tagctccata 9120catgccagat gttcttgaga aactggaatt gctccaaagg aggtttggcg gaacagtgat 9180caggaaccct ctctccagga attccactca tgaaatgtac tacgtgtctg gagcccgcag 9240caatgtcaca tttactgtga accaaacatc ccgcctcctg atgaggagaa tgaggcgtcc 9300aactggaaaa gtgaccctgg aggctgacgt catcctccca attgggacac gcagtgttga 9360gacagacaag ggacccctgg acaaagaggc catagaagaa agggttgaga ggataaaatc 9420tgagtacatg acctcttggt tttatgacaa tgacaacccc tacaggacct ggcactactg 9480tggctcctat gtcacaaaaa cctcaggaag tgcggcgagc atggtaaatg gtgttattaa 9540aattctgaca tatccatggg acaggataga ggaggtcacc agaatggcaa tgactgacac 9600aacccctttt ggacagcaaa gagtgtttaa agaaaaagtt gacaccagag caaaggatcc 9660accagcggga actaggaaga tcatgaaagt tgtcaacagg tggctgttcc gccacctggc 9720cagagaaaag agccccagac tgtgcacaaa ggaagaattt attgcaaaag tccgaagtca 9780tgcagccatt ggagcttacc tggaagaaca agaacagtgg aagactgcca atgaggctgt 9840ccaagaccca aagttctggg aactggtgga tgaagaaagg aagctgcacc aacaaggcag 9900gtgtcggact tgtgtgtaca acatgatggg gaaaagagag aagaagctgt cagagtttgg 9960gaaagcaaag ggaagccgtg ccatatggta tatgtggctg ggagcgcggt atcttgagtt 10020tgaggccctg ggattcctga atgaggacca ttgggcttcc agggaaaact caggaggagg 10080agtggaaggc attggcttac aatacctagg atatgtgatc agagacctgg ctgcaatgga 10140tggtggtgga ttctacgcgg atgacaccgc tggatgggac acgcgcatca cagaggcaga 10200ccttgatgat gaacaggaga tcttgaacta catgagccca catcacaaaa aactggcaca 10260agcagtgatg gaaatgacat acaagaacaa agtggtgaaa gtgttgagac cagccccagg 10320agggaaagcc tacatggatg tcataagtcg acgagaccag agaggatccg ggcaggtagt 10380gacttatgct ctgaacacca tcaccaactt gaaagtccaa ttgatcagaa tggcagaagc 10440agagatggtg atacatcacc aacatgttca agattgtgat gaatcagttc tgaccaggct 10500ggaggcatgg ctcactgagc acggatgtaa cagactgaag aggatggcgg tgagtggaga 10560cgactgtgtg gtccggccca tcgatgacag gttcggcctg gccctgtccc atctcaacgc 10620catgtccaag gttagaaagg acatatctga atggcagcca tcaaaagggt ggaatgattg 10680ggagaatgtg cccttctgtt cccaccactt ccatgaacta cagctgaagg atggcaggag 10740gattgtggtg ccttgccgag aacaggacga gctcattggg agaggaaggg tgtctccagg 10800aaacggctgg atgatcaagg aaacagcttg cctcagcaaa gcctatgcca acatgtggtc 10860actgatgtat tttcacaaaa gggacatgag gctactgtca ttggctgttt cctcagctgt 10920tcccacctca tgggttccac aaggacgcac aacatggtcg attcatggga aaggggagtg 10980gatgaccacg gaagacatgc ttgaggtgtg gaacagagta tggataacca acaacccaca 11040catgcaggac aagacaatgg tgaaaaaatg gagagatgtc ccttatctaa ccaagagaca 11100agacaagctg tgcggatcac tgattggaat gaccaatagg gccacctggg cctcccacat 11160ccatttagtc atccatcgta tccgaacgct gattggacag gagaaataca ctgactacct 11220aacagtcatg gacaggtatt ctgtggatgc tgacctgcaa ctgggtgagc ttatctgaaa 11280caccatctaa caggaataac cgggatacaa accacgggtg gagaaccgga ctccccacaa 11340cctgaaaccg ggatataaac cacggctgga gaaccgggct ccgcacttaa aatgaaacag 11400aaaccgggat aaaaactacg gatggagaac cggactccac acattgagac agaagaagtt 11460gtcagcccag aaccccacac gagttttgcc actgctaagc tgtgaggcag tgcaggctgg 11520gacagccgac ctccaggttg cgaaaaacct ggtttctggg acctcccacc ccagagtaaa 11580aagaacggag cctccgctac caccctccca cgtggtggta gaaagacggg gtctagaggt 11640tagaggagac cctccaggga acaaatagtg ggaccatatt gacgccaggg aaagaccgga 11700gtggttctct gcttttcctc cagaggtctg tgagcacagt ttgctcaaga ataagcagac 11760ctttggatga caaacacaaa accac 1178583718PRTSimian immunodeficiency virus 8Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val Arg1 5 10 15Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys Gln 20 25 30Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile Phe 35 40 45Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His Leu 50 55 60Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val Leu65 70 75 80Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser Ser 85 90 95Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile Leu 100 105 110Gly Met Leu Leu Met Thr Gly Gly Val Thr Leu Val Arg Lys Asn Arg 115 120 125Trp Leu Leu Leu Asn Val Thr Ser Glu Asp Leu Gly Lys Thr Phe Ser 130 135 140Val Gly Thr Gly Asn Cys Thr Thr Asn Ile Leu Glu Ala Lys Tyr Trp145 150 155 160Cys Pro Asp Ser Met Glu Tyr Asn Cys Pro Asn Leu Ser Pro Arg Glu 165 170 175Glu Pro Asp Asp Ile Asp Cys Trp Cys Tyr Gly Val Glu Asn Val Arg 180 185 190Val Ala Tyr Gly Lys Cys Asp Ser Ala Gly Arg Ser Arg Arg Ser Arg 195 200 205Arg Ala Ile Asp Leu Pro Thr His Glu Asn His Gly Leu Lys Thr Arg 210 215 220Gln Glu Lys Trp Met Thr Gly Arg Met Gly Glu Arg Gln Leu Gln Lys225 230 235 240Ile Glu Arg Trp Phe Val Arg Asn Pro Phe Phe Ala Val Thr Ala Leu 245 250 255Thr Ile Ala Tyr Leu Val Gly Ser Asn Met Thr Gln Arg Val Val Ile 260 265 270Ala Leu Leu Val Leu Ala Val Gly Pro Ala Tyr Ser Ala His Cys Ile 275 280 285Gly Ile Thr Asp Arg Asp Phe Ile Glu Gly Val His Gly Gly Thr Trp 290 295 300Val Ser Ala Thr Leu Glu Gln Asp Lys Cys Val Thr Val Met Ala Pro305 310 315 320Asp Lys Pro Ser Leu Asp Ile Ser Leu Glu Thr Val Ala Ile Asp Arg 325 330 335Pro Ala Glu Ala Arg Lys Val Cys Tyr Asn Ala Val Leu Thr His Val 340 345 350Lys Ile Asn Asp Lys Cys Pro Ser Thr Gly Glu Ala His Leu Ala Glu 355 360 365Glu Asn Glu Gly Asp Asn Ala Cys Lys Arg Thr Tyr Ser Asp Arg Gly 370 375 380Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Ile Val Ala Cys385 390 395 400Ala Lys Phe Thr Cys Ala Lys Ser Met Ser Leu Phe Glu Val Asp Gln 405 410 415Thr Lys Ile Gln Tyr Val Ile Arg Ala Gln Leu His Val Gly Ala Lys 420 425 430Gln Glu Asn Trp Asn Thr Ser Ile Lys Thr Leu Lys Phe Asp Ala Leu 435 440 445Ser Gly Ser Gln Glu Val Glu Phe Ile Gly Tyr Gly Lys Ala Thr Leu 450 455 460Glu Cys Gln Val Gln Thr Ala Val Asp Phe Gly Asn Ser Tyr Ile Ala465 470 475 480Glu Met Glu Thr Glu Ser Trp Ile Val Asp Arg Gln Trp Ala Gln Asp 485 490 495Leu Thr Leu Pro Trp Gln Ser Gly Ser Gly Gly Val Trp Arg Glu Met 500 505 510His His Leu Val Glu Phe Glu Pro Pro His Ala Ala Thr Ile Arg Val 515 520 525Leu Ala Leu Gly Asn Gln Glu Gly Ser Leu Lys Thr Ala Leu Thr Gly 530 535 540Ala Met Arg Val Thr Lys Asp Thr Asn Asp Asn Asn Leu Tyr Lys Leu545 550 555 560His Gly Gly His Val Ser Cys Arg Val Lys Leu Ser Ala Leu Thr Leu 565 570 575Lys Gly Thr Ser Tyr Lys Ile Cys Thr Asp Lys Met Phe Phe Val Lys 580 585 590Asn Pro Thr Asp Thr Gly His Gly Thr Val Val Met Gln Val Lys Val 595 600 605Ser Lys Gly Thr Pro Cys Arg Ile Pro Val Ile Val Ala Asp Asp Leu 610 615 620Thr Ala Ala Ile Asn Lys Gly Ile Leu Val Thr Val Asn Pro Ile Ala625 630 635 640Ser Thr Asn Asp Asp Glu Val Leu Ile Glu Val Asn Pro Pro Phe Gly 645 650 655Asp Ser Tyr Ile Ile Val Gly Arg Gly Asp Ser Arg Leu Thr Tyr Gln 660 665 670Trp His Lys Glu Gly Ser Ser Ile Gly Lys Leu Phe Thr Gln Thr Met 675 680 685Lys Gly Val Glu Arg Leu Ala Val Met Gly Asp Thr Ala Trp Asp Phe 690 695 700Ser Ser Ala Gly Gly Phe Phe Thr Ser Val Gly Lys Gly Ile His Thr705 710 715 720Val Phe Gly Ser Ala Phe Gln Gly Leu Phe Gly Gly Leu Asn Trp Ile 725 730 735Thr Lys Val Ile Met Gly Ala Val Leu Ile Trp Val Gly Ile Asn Thr 740 745 750Arg Asn Met Thr Met Ser Met Ser Met Ile Leu Val Gly Val Ile Met 755 760 765Met Phe Leu Ser Leu Gly Val Gly Ala Asp Gln Gly Ser Ala Ile Asn 770 775 780Phe Gly Arg Gly Leu Ala Glu Ser Leu Leu Glu Asn Lys Glu Gly Cys785 790 795 800Gln Lys Ile Leu Ser Val Leu Ala Pro Leu Val Pro Thr Gly Ser Glu 805 810 815Asn Leu Lys Ser Leu Tyr Asn Thr Val Cys Val Ile Trp Cys Ile His 820 825 830Ala Glu Glu Lys Val Lys His Thr Glu Glu Ala Lys Gln Ile Val Gln 835 840 845Arg His Leu Val Val Glu Thr Gly Thr Thr Glu Thr Met Pro Lys Thr 850 855 860Ser Arg Pro Thr Ala Pro Ser Ser Gly Arg Gly Gly Asn Tyr Pro Val865 870 875 880Gln Gln Ile Gly Gly Asn Tyr Val His Leu Pro Leu Ser Pro Arg Thr 885 890 895Leu Asn Ala Trp Val Lys Leu Ile Glu Glu Lys Lys Phe Gly Ala Glu 900 905 910Val Val Pro Gly Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro Tyr Asp 915 920 925Ile Asn Gln Met Leu Asn Cys Val Gly Asp His Gln Ala Ala Met Gln 930 935 940Ile Ile Arg Asp Ile Ile Asn Glu Glu Ala Ala Asp Trp Asp Leu Gln945 950 955 960His Pro Gln Pro Ala Pro Gln Gln Gly Gln Leu Arg Glu Pro Ser Gly 965 970 975Ser Asp Ile Ala Gly Thr Thr Ser Ser Val Asp Glu Gln Ile Gln Trp 980 985 990Met Tyr Arg Gln Gln Asn Pro Ile Pro Val Gly Asn Ile Tyr Arg Arg 995 1000 1005Trp Ile Gln Leu Lys Glu Gly Ser Ser Ile Gly Thr Ser Leu Gly 1010 1015 1020Lys Ala Val His Gln Val Phe Gly Ser Val Tyr Thr Thr Met Phe 1025 1030 1035Gly Gly Val Ser Trp Met Ile Arg Ile Leu Ile Gly Phe Leu Val 1040 1045 1050Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr Ser Met Ala Met Thr 1055 1060 1065Cys Ile Ala Val Gly Gly Ile Thr Leu Phe Leu Gly Phe Thr Val 1070 1075 1080Gly Ala Asp Gln Gly Cys Ala Ile Asn Phe Gly Lys Arg Glu Leu 1085 1090 1095Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg Asp Ser Asp Asp Trp 1100 1105 1110Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro Val Lys Leu Ala 1115 1120 1125Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys Gly Leu Asn 1130 1135 1140Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg Ala Asp 1145 1150 1155Glu Ile Asn Thr Ile Phe Glu Glu Asn Glu Val Asp Ile Ser Val 1160 1165 1170Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro 1175 1180 1185Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp 1190 1195 1200Gly Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe 1205 1210 1215Ile Ile Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg 1220 1225 1230Val Trp Asn Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe 1235 1240 1245Thr Thr Arg Val Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp 1250 1255 1260Cys Asp Gly Ser Ile Leu Gly Ala Ala Val Asn Gly Lys Lys Ser 1265 1270 1275Ala His Gly Ser Pro Thr Phe Trp Met Gly Ser His Glu Val Asn 1280 1285 1290Gly Thr Trp Met Ile His Thr Leu Glu Ala Leu Asp Tyr Lys Glu 1295 1300 1305Cys Glu Trp Pro Leu Thr His Thr Ile Gly Thr Ser Val Glu Glu 1310 1315 1320Ser Glu Met Phe Met Pro Arg Ser Ile Gly Gly Pro Val Ser Ser 1325 1330 1335His Asn His Ile Pro Gly Tyr Lys Val Gln Thr Asn Gly Pro Trp 1340 1345 1350Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala Cys Pro Gly Thr 1355 1360 1365Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg Gly Lys Ser Thr 1370 1375 1380Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu Trp Cys Cys 1385 1390 1395Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser Asp Gly 1400 1405 1410Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser 1415 1420 1425His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val 1430 1435 1440Pro Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu 1445 1450 1455Arg Lys Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val 1460 1465 1470Leu Leu Gly Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu 1475 1480 1485Leu Lys Leu Thr Val Ala Val Gly Leu His Phe His Glu Met Asn 1490 1495 1500Asn Gly Gly Asp Ala Met Tyr Met Ala Leu Ile Ala Ala Phe Ser 1505 1510 1515Ile Arg Pro Gly Leu Leu Ile Gly Phe Gly Leu Arg Thr Leu Trp 1520 1525 1530Ser Pro Arg Glu Arg Leu Val Leu Thr Leu Gly Ala Ala Met Val 1535 1540 1545Glu Ile Ala Leu Gly Gly Val Met Gly Gly Leu Trp Lys Tyr Leu 1550 1555 1560Asn Ala Val Ser Leu Cys Ile Leu Thr Ile Asn Ala Val Ala Ser 1565 1570 1575Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu Met Ala Leu Leu Thr 1580 1585 1590Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala Met Phe Phe Cys 1595 1600 1605Ala Met Val Ile Ile Gly Val Leu His Gln Asn Phe Lys Asp Thr 1610 1615 1620Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr Leu Thr Ser 1625 1630 1635Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala Phe Leu 1640 1645 1650Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala 1655 1660 1665Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe 1670 1675 1680Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu 1685 1690 1695Leu Met Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu 1700 1705 1710Leu Lys Lys Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile 1715 1720 1725Ser Gly Ser Ser Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly 1730 1735 1740Glu Phe Lys Leu Leu Ser Glu Glu Lys Val Pro Trp Asp Gln Val 1745 1750 1755Val Met Thr Ser Leu Ala Leu Val Gly Ala Ala Leu His Pro Phe 1760 1765 1770Ala Leu Leu Leu Val Leu Ala Gly Trp Leu Phe His Val Arg Gly 1775 1780 1785Ala Arg Arg Ser Gly Asp Val Leu Trp Asp Ile Pro Thr Pro Lys 1790 1795 1800Ile Ile Glu Glu Cys Glu His Leu Glu Asp Gly Ile Tyr Gly Ile 1805 1810 1815Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln Arg Gly Val Gly Val 1820 1825 1830Ala Gln Gly Gly Val Phe His Thr Met Trp His Val Thr Arg Gly 1835 1840 1845Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile Pro Ser Trp Ala 1850 1855 1860Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser Trp Lys Leu 1865 1870 1875Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile Ala Ala 1880 1885

1890Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu 1895 1900 1905Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp 1910 1915 1920Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly 1925 1930 1935Glu Val Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn 1940 1945 1950Ser Phe Val Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly 1955 1960 1965Lys Glu Glu Leu Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met 1970 1975 1980Thr Thr Val Leu Asp Phe His Pro Gly Ala Gly Lys Thr Arg Arg 1985 1990 1995Phe Leu Pro Gln Ile Leu Ala Glu Cys Ala Arg Arg Arg Leu Arg 2000 2005 2010Thr Leu Val Leu Ala Pro Thr Arg Val Val Leu Ser Glu Met Lys 2015 2020 2025Glu Ala Phe His Gly Leu Asp Val Lys Phe His Thr Gln Ala Phe 2030 2035 2040Ser Ala His Gly Ser Gly Arg Glu Val Ile Asp Ala Met Cys His 2045 2050 2055Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro Thr Arg Val Val Asn 2060 2065 2070Trp Glu Val Ile Ile Met Asp Glu Ala His Phe Leu Asp Pro Ala 2075 2080 2085Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg Ala Arg Ala Asn 2090 2095 2100Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro Gly Thr Ser 2105 2110 2115Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln Thr 2120 2125 2130Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu 2135 2140 2145Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala 2150 2155 2160Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val 2165 2170 2175Val Val Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile 2180 2185 2190Lys Gln Lys Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu 2195 2200 2205Met Gly Ala Asn Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr 2210 2215 2220Ala Phe Lys Pro Val Leu Val Asp Glu Gly Arg Lys Val Ala Ile 2225 2230 2235Lys Gly Pro Leu Arg Ile Ser Ala Ser Ser Ala Ala Gln Arg Arg 2240 2245 2250Gly Arg Ile Gly Arg Asn Pro Asn Arg Asp Gly Asp Ser Tyr Tyr 2255 2260 2265Tyr Ser Glu Pro Thr Ser Glu Asn Asn Ala His His Val Cys Trp 2270 2275 2280Leu Glu Ala Ser Met Leu Leu Asp Asn Met Glu Val Arg Gly Gly 2285 2290 2295Met Val Ala Pro Leu Tyr Gly Val Glu Gly Thr Lys Thr Pro Val 2300 2305 2310Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln Arg Lys Val Phe 2315 2320 2325Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val Trp Leu Ser Trp 2330 2335 2340Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys Trp Cys 2345 2350 2355Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly Glu 2360 2365 2370Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg 2375 2380 2385Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu 2390 2395 2400Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu 2405 2410 2415Val Leu Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys 2420 2425 2430Gly Gly Glu Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu 2435 2440 2445Glu Gly Ser Arg Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu 2450 2455 2460Ala Met Thr Ile Val Met Leu Phe Ile Leu Ala Gly Leu Leu Thr 2465 2470 2475Ser Gly Met Val Ile Phe Phe Met Ser Pro Lys Gly Ile Ser Arg 2480 2485 2490Met Ser Met Ala Met Gly Thr Met Ala Gly Cys Gly Tyr Leu Met 2495 2500 2505Phe Leu Gly Gly Val Lys Pro Thr His Ile Ser Tyr Val Met Leu 2510 2515 2520Ile Phe Phe Val Leu Met Val Val Val Ile Pro Glu Pro Gly Gln 2525 2530 2535Gln Arg Ser Ile Gln Asp Asn Gln Val Ala Tyr Leu Ile Ile Gly 2540 2545 2550Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn Glu Leu Gly Met 2555 2560 2565Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys Lys Asn Leu Ile 2570 2575 2580Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp Leu Lys 2585 2590 2595Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met Leu 2600 2605 2610Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu 2615 2620 2625Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met 2630 2635 2640Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met 2645 2650 2655Leu Leu Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu 2660 2665 2670Cys Gly Ile Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro 2675 2680 2685Gly Ile Lys Ala Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe 2690 2695 2700His Gly Val Ala Lys Asn Pro Val Val Asp Gly Asn Pro Thr Val 2705 2710 2715Asp Ile Glu Glu Ala Pro Glu Met Pro Ala Leu Tyr Glu Lys Lys 2720 2725 2730Leu Ala Leu Tyr Leu Leu Leu Ala Leu Ser Leu Ala Ser Val Ala 2735 2740 2745Met Cys Arg Thr Pro Phe Ser Leu Ala Glu Gly Ile Val Leu Ala 2750 2755 2760Ser Ala Ala Leu Gly Pro Leu Ile Glu Gly Asn Thr Ser Leu Leu 2765 2770 2775Trp Asn Gly Pro Met Ala Val Ser Met Thr Gly Val Met Arg Gly 2780 2785 2790Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn Leu Trp Lys Met 2795 2800 2805Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys Thr Leu Gly Glu 2810 2815 2820Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln Phe Glu 2825 2830 2835Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr Ala 2840 2845 2850Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val 2855 2860 2865Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr 2870 2875 2880Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly 2885 2890 2895Gly Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val 2900 2905 2910Lys Gly Phe Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn 2915 2920 2925Val Gln Ser Leu Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr 2930 2935 2940Asp Ile His Arg Leu Glu Pro Val Lys Cys Asp Thr Leu Leu Cys 2945 2950 2955Asp Ile Gly Glu Ser Ser Ser Ser Ser Val Thr Glu Gly Glu Arg 2960 2965 2970Thr Val Arg Val Leu Asp Thr Val Glu Lys Trp Leu Ala Cys Gly 2975 2980 2985Val Asp Asn Phe Cys Val Lys Val Leu Ala Pro Tyr Met Pro Asp 2990 2995 3000Val Leu Glu Lys Leu Glu Leu Leu Gln Arg Arg Phe Gly Gly Thr 3005 3010 3015Val Ile Arg Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr 3020 3025 3030Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe Thr Val Asn Gln 3035 3040 3045Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg Pro Thr Gly Lys 3050 3055 3060Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly Thr Arg Ser 3065 3070 3075Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu Glu 3080 3085 3090Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr 3095 3100 3105Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr 3110 3115 3120Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val 3125 3130 3135Ile Lys Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr 3140 3145 3150Arg Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val 3155 3160 3165Phe Lys Glu Lys Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly 3170 3175 3180Thr Arg Lys Ile Met Lys Val Val Asn Arg Trp Leu Phe Arg His 3185 3190 3195Leu Ala Arg Glu Lys Ser Pro Arg Leu Cys Thr Lys Glu Glu Phe 3200 3205 3210Ile Ala Lys Val Arg Ser His Ala Ala Ile Gly Ala Tyr Leu Glu 3215 3220 3225Glu Gln Glu Gln Trp Lys Thr Ala Asn Glu Ala Val Gln Asp Pro 3230 3235 3240Lys Phe Trp Glu Leu Val Asp Glu Glu Arg Lys Leu His Gln Gln 3245 3250 3255Gly Arg Cys Arg Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu 3260 3265 3270Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile 3275 3280 3285Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe Glu Ala Leu 3290 3295 3300Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu Asn Ser Gly 3305 3310 3315Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val Ile 3320 3325 3330Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp 3335 3340 3345Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp 3350 3355 3360Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu 3365 3370 3375Ala Gln Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys 3380 3385 3390Val Leu Arg Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile 3395 3400 3405Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala 3410 3415 3420Leu Asn Thr Ile Thr Asn Leu Lys Val Gln Leu Ile Arg Met Ala 3425 3430 3435Glu Ala Glu Met Val Ile His His Gln His Val Gln Asp Cys Asp 3440 3445 3450Glu Ser Val Leu Thr Arg Leu Glu Ala Trp Leu Thr Glu His Gly 3455 3460 3465Cys Asn Arg Leu Lys Arg Met Ala Val Ser Gly Asp Asp Cys Val 3470 3475 3480Val Arg Pro Ile Asp Asp Arg Phe Gly Leu Ala Leu Ser His Leu 3485 3490 3495Asn Ala Met Ser Lys Val Arg Lys Asp Ile Ser Glu Trp Gln Pro 3500 3505 3510Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro Phe Cys Ser His 3515 3520 3525His Phe His Glu Leu Gln Leu Lys Asp Gly Arg Arg Ile Val Val 3530 3535 3540Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg Val Ser 3545 3550 3555Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser Lys 3560 3565 3570Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp 3575 3580 3585Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser 3590 3595 3600Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly 3605 3610 3615Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val 3620 3625 3630Trp Ile Thr Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys 3635 3640 3645Lys Trp Arg Asp Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu 3650 3655 3660Cys Gly Ser Leu Ile Gly Met Thr Asn Arg Ala Thr Trp Ala Ser 3665 3670 3675His Ile His Leu Val Ile His Arg Ile Arg Thr Leu Ile Gly Gln 3680 3685 3690Glu Lys Tyr Thr Asp Tyr Leu Thr Val Met Asp Arg Tyr Ser Val 3695 3700 3705Asp Ala Asp Leu Gln Leu Gly Glu Leu Ile 3710 37159508PRTHuman immunodeficiency virus 9Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Lys Trp1 5 10 15Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Thr Tyr Gln Leu Lys 20 25 30His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45Gly Leu Leu Glu Thr Gly Gly Gly Cys Lys Gln Ile Leu Val Gln Leu 50 55 60Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Lys Ser Leu Tyr Asn65 70 75 80Ala Val Ala Thr Leu Tyr Cys Val His Gln Gly Ile Glu Val Arg Asp 85 90 95Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys 100 105 110Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gly Asn Ser Ser Gln Val 115 120 125Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His 130 135 140Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu145 150 155 160Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser 165 170 175Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly 180 185 190Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu 195 200 205Ala Ala Glu Trp Asp Arg Leu His Pro Ala His Ala Gly Pro Asn Ala 210 215 220Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr225 230 235 240Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Ser Asn Pro Pro Val 245 250 255Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 260 265 270Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly 275 280 285Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 290 295 300Arg Ala Glu Gln Ala Ser Gln Asp Val Lys Asn Trp Met Thr Glu Thr305 310 315 320Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala 325 330 335Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly 340 345 350Val Gly Gly Pro Ser His Lys Ala Arg Ile Leu Ala Glu Ala Met Ser 355 360 365Gln Val Thr Ser Pro Ala Asn Ile Met Met Gln Arg Gly Asn Phe Arg 370 375 380Asn Gln Arg Lys Thr Ile Lys Cys Phe Asn Cys Gly Lys Glu Gly His385 390 395 400Leu Ala Arg His Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys 405 410 415Gly Arg Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn 420 425 430Phe Leu Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe 435 440 445Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg 450 455 460Phe Gly Glu Glu Thr Thr Thr Pro Pro Gln Lys Gln Glu Pro Leu Pro465 470 475 480Ser Gln Lys Gln Glu Thr Ile Asp Lys Asp Leu Tyr Pro Leu Ala Ser 485 490 495Leu Lys Ser Leu Phe Gly Asn Asp Pro Ser Leu Gln 500 505

Claims

1. A pharmaceutical composition for infection caused by lentiviruses, comprising: (A) a recombinant yellow fever virus which expresses a heterologous polypeptide comprising at least a fragment of a lentiviral polypeptide, and (B) a pharmaceutical carrier; wherein the composition comprises the recombinant yellow fever virus in an amount effective to induce a therapeutic or protective immune response against infection with lentivirus.

2. The composition according to claim 1, wherein the heterologous polypeptide includes at least a fragment of an HIV Gag polypeptide or at least a fragment of athe polypeptide of SIV Gag.

3. The composition according to claim 1, wherein the heterologous polypeptide includes at least a fragment of SEQ ID NO:9.

4. The composition according to claim 1, wherein the fragment comprises at least 8, 9, 10, 20, 30, 40, 50, 100 or 200 continuous amino acids continuous of SEQ ID NO:9.

5. The composition according to claim 1, wherein the fragment comprises amino acids 45-269 of SEQ ID NO:9.

6. The composition according to claim 1, wherein the heterologous polypeptide comprises a sequence of amino acid C-terminal comprising KESSIG.

7. The composition according to claim 1, wherein the recombinant yellow fever virus is YF17D.

8. The composition according to claim 1, wherein the recombinant yellow fever virus comprises a sequence coding for the heterologous polypeptide inserted between sequences coding for polypeptide and NS1 polypeptide.

9. The composition according to claim 1 characterized in that the recombinant yellow fever virus comprises a sequence coding for the heterologous polypeptide, said sequence being optimized according to codon usage frequency of the yellow fever virus.

10. A pharmaceutical composition for infection caused by lentiviruses, comprising a mixture of recombinant yellow fever viruses, wherein the mixture comprises: (A) a first recombinant yellow fever virus which expresses a first heterologous polypeptide comprising at least a fragment of a lentiviral Gag polypeptide; (B) a second recombinant yellow fever virus which expresses a second heterologous polypeptide comprising at least a fragment of a lentiviral Vif polypeptide; (C) a third recombinant yellow fever virus which expresses a third heterologous polypeptide comprising at least a fragment of a lentiviral Nef polypeptide, and (D) a pharmaceutical carrier, wherein the composition comprises the mixture in an amount effective to induce a protective or therapeutic immune response against infection lentiviral.

11. The composition according to claim 10, wherein the Gag polypeptide is a Gag polypeptide of HIV; the Vif polypeptide is a Vif polypeptide of HIV; and the Nef polypeptide is a Nef polypeptide of HIV.

12. A method for inducing a protective or therapeutic immune response against HIV, wherein the method comprises administering the composition defined in claim 1 to an individual who needs it.

13. The method according to claim 12, further comprising administering an initial dose of the composition of claim 1 in a priming step prior to administering the composition of claim 1, wherein the initial dose comprises a DNA encoding one or more of HIV polypeptides or fragments thereof and/or the initial dosage of the composition comprises rBCG that expresses one or more of polypeptides of HIV or fragments thereof.

14. The method according to claim 12, wherein the composition is administered in two or more subsequent times, waiting for at least 4-12 weeks before the subsequent administration.

15. The method according to claim 12, wherein a protective or therapeutic immune response against HIV includes a response of CD4+ T cell and/or a response in CD8+ Tcell.

16. A vaccine to induce a protective immune response or a therapeutic response against HIV, wherein said vaccine comprises: (A) a recombinant yellow fever virus which expresses a heterologous polypeptide comprising at least a fragment of a lentiviral polypeptide; (B) a pharmaceutical carrier; wherein the composition comprises the recombinant yellow fever virus in an amount effective to induce a therapeutic or protective immune response against infection with lentivirus.

17. A vaccine to induce a protective immune response or therapeutic response against HIV, wherein said vaccine comprises a mixture of recombinant yellow fever virus, said mixture comprising: (A) a first recombinant yellow fever virus which expresses a first heterologous polypeptide comprising at least a fragment of a lentiviral Gag polypeptide; (B) a second recombinant yellow fever virus expressing a second heterologous polypeptide comprising at least a fragment of a lentiviral Vif polypeptide; (C) a third recombinant yellow fever virus that expresses a third heterologous polypeptide comprising at least a fragment of a lentiviral Nef polypeptide; and (D) a pharmaceutical carrier.

18. A 45-269 amino acid fragment of the Gag protein used for drawing a heterologous cassette of SIV, wherein the fragment comprises SEQ ID NO:1.

19. A recombinant protein of 45-269 amino acids from Gag protein, wherein the recombinant protein comprises SEQ ID NO:2.

20. A recombinant gene of Gag 45-269 protein of SIV, wherein the gene comprises SEQ ID NO:6.

21. A nucleotide sequence of recombinant yellow fever virus which expresses Gag 45-269 protein, wherein the nucleotide sequence comprises SEQ ID NO:7.

22. A precursor polyprotein containing a recombinant protein of amino acids 45-269 from the SIV Gag protein, wherein the precursor polyprotein comprises SEQ ID NO:8.

23. A method for producing lentivirus proteins or related polypeptides, wherein said method comprises the steps of: (A) introducing at least one vector or a vector system in a host cell, said vector or vector system including a nucleic acid sequence encoding at least an immunogenic protein of lentivirus or a related polypeptide, a nucleic acid sequence having at least 90% identity with sequence SEQ ID NO:6 or SEQ ID NO:7 or an encoded protein having at least 90% identity with a protein defined by SEQ ID NOs:1, 2, 8 or 9; (B) expressing the nucleic acid sequence into the host cell; and (C) producing anthe antigenic protein encoded by lentivirus or a related polypeptide within the host cell.

24. The method according to claim 23, wherein two or more vectors or vector systems are introduced.

25. The method according to claim 23, wherein the vector or vector system includes a nucleic acid sequence encoding two or more immunogenic proteins of lentiviruses or related polypeptides.